Secondary literature sources for S_TKc

The following references were automatically generated.

Burk DL, Hon WC, Leung AK, Berghuis AM
Structural Analyses of Nucleotide Binding to an Aminoglycoside Phosphotransferase(,).
Biochemistry. 2001; 40: 8756-64
Display abstract
3',5' '-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.

Haq R, Randall S, Midmer M, Yee K, Zanke B
NKIATRE is a novel conserved cdc2-related kinase.
Genomics. 2001; 71: 131-41
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The mitogen-activated protein kinases (MAPKs) and the cyclin-dependent kinases (CDKs) are key mediators of cell proliferation in response to extracellular signals. Recent additions to each of these families and the identification of kinases with structural features of both have provided insights into fundamental processes, such as cell division and differentiation. To identify novel serine kinases with features of MAPKs or CDKs, a degenerate PCR-based amplification approach was undertaken. The 57- and 52-kDa isoforms of a novel protein kinase, termed NKIATRE, were molecularly cloned from rat brain and jejunum cDNA libraries. Like the MAPKs, NKIATRE has a Thr-Xaa-Tyr motif in kinase subdomain VIII. NKIATRE also shows close homology to the cyclin-dependent kinase class of protein kinases and the cdc2-related kinases NKIAMRE, KKIALRE, and KKIAMRE, containing both conserved inhibitory phosphorylation sites and a putative cyclin-binding domain. Two isoforms of NKIATRE that differ in their carboxy-terminal ends have been identified. A functional nuclear localization signal is specific to the longer 57-kDa alpha isoform. Sequence similarity to the putative human tumor suppressor gene NKIAMRE, which is lost in leukemic patients with chromosome 5q deletions, suggests that NKIATRE may have a role in restricting cell growth or maintaining differentiation. Copyright 2001 Academic Press.

Parang K, Till JH, Ablooglu AJ, Kohanski RA, Hubbard SR, Cole PA
Mechanism-based design of a protein kinase inhibitor.
Nat Struct Biol. 2001; 8: 37-41
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Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPgammaS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors.

Sielecki TM et al.
Quinazolines as cyclin dependent kinase inhibitors.
Bioorg Med Chem Lett. 2001; 11: 1157-60
Display abstract
Quinazolines have been identified as inhibitors of CDK4/D1 and CDK2/E. Aspects of the SAR were investigated using solution-phase, parallel synthesis. An X-ray crystal structure was obtained of quinazoline 51 bound in CDK2 and key interactions within the ATP binding pocket are defined.

Tholey A, Pipkorn R, Bossemeyer D, Kinzel V, Reed J
Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase.
Biochemistry. 2001; 40: 225-31
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A number of isoenzymes of the catalytic subunit of cAMP-dependent protein kinase arise through posttranslational modifications of the enzyme outside the catalytic domain; the biological significance of these is not yet fully clear. A clustering of sites for such modification exists at the N-terminus of the protein, where myristoylation (of Gly1), phosphorylation (at Ser10), and deamidation of Asn2 have been observed. As the first two are known to govern membrane binding and thus subcellular compartmentalization in some proteins, it was of interest to see whether the local structure of the N-terminus was being influenced by one or more of these modifications. A series of synthetic peptides mimicing the 16 N-terminal residues of the catalytic subunit Calpha was produced covering the full range of possible modifications, singly and in combination, and tested for possible effects on local structure by measuring the circular dichroism under varying polarity. It was found that myristoylation and phosphorylation modify the structure in this region in opposite ways and in a manner designed to amplify the action of a potential myristoyl/electrostatic switch. To what extent deamidation of Asn2 may oppose a potential membrane binding is unknown. Deamidation, however, had no effect on the structure of the peptide either alone or in combination with acylation and/or phosphorylation, suggesting that the change of the nuclear/cytoplasmic disribution in cells caused by deamidation [Pepperkok et al. (2000) J. Cell Biol. 148, 715-726] is due to a more complex signaling mechanism. The structural implications of the data are discussed.

Davies TG et al.
Inhibitor binding to active and inactive cdk2. the crystal structure of cdk2-cyclin a/indirubin-5-sulphonate.
Structure (Camb). 2001; 9: 389-97
Display abstract
Background: Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2.Results: Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten.Conclusions: The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.

Hoofnagle AN, Resing KA, Goldsmith EJ, Ahn NG
Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange.
Proc Natl Acad Sci U S A. 2001; 98: 956-61
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Changes in protein mobility accompany changes in conformation during the trans-activation of enzymes; however, few studies exist that validate or characterize this behavior. In this study, amide hydrogen/deuterium exchange/mass spectrometry was used to probe the conformational flexibility of extracellular signal-regulated protein kinase-2 before and after activation by phosphorylation. The exchange data indicated that extracellular regulated protein kinase-2 activation caused altered backbone flexibility in addition to the conformational changes previously established by x-ray crystallography. The changes in flexibility occurred in regions involved in substrate binding and turnover, suggesting their importance in enzyme regulation.

Dreyer MK et al.
Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717.
J Med Chem. 2001; 44: 524-30
Display abstract
Cyclin-dependent kinases (CDKs) are regulatory proteins of the eukaryotic cell cycle. They act after association with different cyclins, the concentrations of which vary throughout the progression of the cell cycle. As central mediators of cell growth, CDKs are potential targets for inhibitory molecules that would allow disruption of the cell cycle in order to evoke an antiproliferative effect and may therefore be useful as cancer therapeutics. We synthesized several inhibitory 2,6,9-trisubstituted purine derivatives and solved the crystal structure of one of these compounds, H717, in complex with human CDK2 at 2.6 A resolution. The orientation of the C2-p-diaminocyclohexyl portion of the inhibitor is strikingly different from those of similar moieties in other related inhibitor complexes. The N9-cyclopentyl ring fully occupies a space in the enzyme which is otherwise empty, while the C6-N-aminobenzyl substituent points out of the ATP-binding site. The structure provides a basis for the further development of more potent inhibitory drugs.

Bilwes AM, Quezada CM, Croal LR, Crane BR, Simon MI
Nucleotide binding by the histidine kinase CheA.
Nat Struct Biol. 2001; 8: 353-60
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To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg(2+) or Mn(2+). The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide gamma-phosphate and its associated Mg(2+) ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.

Wong CF et al.
Computational analysis of PKA-balanol interactions.
J Med Chem. 2001; 44: 1530-9
Display abstract
Protein kinases are important targets for designing therapeutic drugs. This paper illustrates a computational approach to extend the usefulness of a single protein-inhibitor structure in aiding the design of protein kinase inhibitors. Using the complex structure of the catalytic subunit of PKA (cPKA) and balanol as a guide, we have analyzed and compared the distribution of amino acid types near the protein-ligand interface for nearly 400 kinases. This analysis has identified a number of sites that are more variable in amino acid types among the kinases analyzed, and these are useful sites to consider in designing specific protein kinase inhibitors. On the other hand, we have found kinases whose protein-ligand interfaces are similar to that of the cPKA-balanol complex and balanol can be a useful lead compound for developing effective inhibitors for these kinases. Generally, this approach can help us discover new drug targets for an existing class of compounds that have already been well characterized pharmacologically. The relative significance of the charge/polarity of residues at the protein-ligand interface has been quantified by carrying out computational sensitivity analysis in which the charge/polarity of an atom or functional group was turned off/on, and the resulting effects on binding affinity have been examined. The binding affinity was estimated by using an implicit-solvent model in which the electrostatic contributions were obtained by solving the Poisson equation and the hydrophobic effects were accounted for by using surface-area dependent terms. The same sensitivity analysis approach was applied to the ligand balanol to develop a pharmacophoric model for searching new drug leads from small-molecule libraries. To help evaluate the binding affinity of designed inhibitors before they are made, we have developed a semiempirical approach to improve the predictive reliability of the implicit-solvent binding model.

Wang J et al.
Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.
Structure (Camb). 2001; 9: 177-84
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BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

Zhou B, Wu L, Shen K, Zhang J, Lawrence DS, Zhang ZY
Multiple regions of MAP kinase phosphatase 3 are involved in its recognition and activation by ERK2.
J Biol Chem. 2001; 276: 6506-15
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Mitogen-activated protein kinase phosphatase 3 (MKP3) is a specific regulator of extracellular signal-regulated protein kinase 2 (ERK2). Association of ERK2 with MKP3 results in a powerful increase in MKP3 phosphatase activity. To determine the molecular basis of the specific ERK2 recognition by MKP3 and the ERK2-induced MKP3 activation, we have carried out a systematic mutational and deletion analysis of MKP3. Using activation-based and competition-based assays, we are able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP3 activation. Our results show that recognition and activation of MKP3 by ERK2 involves multiple regions of MKP3. Thus, the kinase interaction motif (KIM; residues 61--75) in MKP3 plays a major role (135-fold) for high affinity ERK2 binding. The most important residue in the KIM sequence of MKP3 is Arg(65), which probably interacts with Asp(319) in ERK2. In addition to KIM, a unique sequence conserved in cytosolic MKPs (residues 161--177 in MKP3) also contributes to ERK2 binding (15-fold). However, these two regions are not essential for ERK2-induced MKP3 activation. A third ERK2 binding site is localized in the C terminus of MKP3 (residues 348--381). Although deletion of this region or mutation of the putative ERK specific docking sequence (364)FTAP(367) in this region reduces MKP3's affinity for ERK2 by less than 10-fold, this region is absolutely required for ERK2-induced MKP3 activation.

Yam CH, Siu WY, Lau A, Poon RY
Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2.
J Biol Chem. 2000; 275: 3158-67
Display abstract
Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

Adkins JN, Lumb KJ
Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1.
Biochemistry. 2000; 39: 13925-30
Display abstract
Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.

Zaremberg V, Donella-Deana A, Moreno S
Mechanism of activation of cAMP-dependent protein kinase: in Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit.
Arch Biochem Biophys. 2000; 381: 74-82
Display abstract
Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.

Shewchuk L et al.
Binding mode of the 4-anilinoquinazoline class of protein kinase inhibitor: X-ray crystallographic studies of 4-anilinoquinazolines bound to cyclin-dependent kinase 2 and p38 kinase.
J Med Chem. 2000; 43: 133-8
Display abstract
4-Anilinoquinazolines represent an important class of protein kinase inhibitor. Modes of binding for two members of this inhibitor class were determined by X-ray crystallographic analysis of one inhibitor (4-[3-hydroxyanilino]-6,7-dimethoxyquinazoline) in complex with cyclin-dependent kinase 2 (CDK2) and the other (4-[3-methylsulfanylanilino]-6,7-dimethoxyquinazoline) in complex with p38 kinase. In both inhibitor/kinase structures, the 4-anilinoquinazoline was bound in the ATP site with the quinazoline ring system oriented along the peptide strand that links the two domains of the protein and with the anilino substituent projecting into a hydrophobic pocket within the protein interior. In each case, the nitrogen at position-1 of the quinazoline accepted a hydrogen bond from a backbone NH (CDK2, Leu-83; p38, Met-109) of the domain connector strand, and aromatic hydrogen atoms at C2 and C8 interacted with backbone carbonyl oxygen atoms of the peptide strand. The anilino group of the CDK2-bound compound was essentially coplanar with the quinazoline ring system and occupied a pocket between Lys-33 and Phe-80. For the p38-bound inhibitor, the anilino group was angled out of plane and was positioned between Lys-53 and Thr-106 in a manner similar to that observed for the aryl substituent of the pyridinylimidazole class of inhibitor.

Means AR
Regulatory cascades involving calmodulin-dependent protein kinases.
Mol Endocrinol. 2000; 14: 4-13

Eisenmesser EZ, Zabell AP, Post CB
Accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data: a simulation study.
J Biomol NMR. 2000; 17: 17-32
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The exchange-transferred NOE method to determine the three-dimensional structure of peptides bound to proteins, or other macromolecular systems, is becoming increasingly important in drug design efforts and for large or multicomponent assemblies, such as membrane receptors, where structural analysis of the full system is intractable. The exchange-transferred nuclear Overhauser effect spectroscopy (etNOESY) method allows the determination of the bound-state conformation of the peptide from the intra-molecular NOE interactions between ligand protons. Because only ligand-ligand NOEs are generally observable, the etNOESY method is restricted to fewer NOEs per residue than direct protein structure determination. In addition, the averaging of relaxation rates between free and bound states affects the measured cross-peak intensities, and possibly the accuracy of distance estimates. Accordingly, the study reported here was conducted to examine the conditions required to define a reliable structure. The program CORONA was used to simulate etNOE data using a rate-matrix including magnetic relaxation and exchange rates for two peptide-protein complexes derived from the reference complex of cAMP-dependent protein kinase ligated with a 24-residue inhibitor peptide. The results indicate that reasonably accurate peptide structures can be determined with relatively few NOE interactions when the interactions occur between non-neighboring residues. The reliability of the structural result is suggested from the pattern of NOE interactions. A structure with an accuracy of approximately 1.3 A rms difference for the main-chain atoms can be obtained when etNOE interactions between non-neighboring residues occur over the length of the peptide. The global precision is higher (approximately 0.9 A rms difference) but is not correlated to global accuracy. A local definition of precision along the backbone appears to be a good indicator of the local accuracy.

Gitig DM, Koff A
Cdk pathway: cyclin-dependent kinases and cyclin-dependent kinase inhibitors.
Methods Mol Biol. 2000; 142: 109-23

Sielecki TM, Boylan JF, Benfield PA, Trainor GL
Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation.
J Med Chem. 2000; 43: 1-18

Diller TC, Xuong NH, Taylor SS
Type II beta regulatory subunit of cAMP-dependent protein kinase: purification strategies to optimize crystallization.
Protein Expr Purif. 2000; 20: 357-64
Display abstract
To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RII beta isoform and five RII beta deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RII beta deletion mutant (delta 1-111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 A resolution were collected at 29 degrees C using synchrotron radiation on a single crystal measuring 0.2 x 0.3 x 1.2 mm(3). Data were 99% complete. The hexagonal crystal belonged to space group P6((1)) or P6((5)) with unit cell dimensions a = b = 161.62 A and c = 39.66 A.

Ducrot P, Legraverend M, Grierson DS
3D-QSAR CoMFA on cyclin-dependent kinase inhibitors.
J Med Chem. 2000; 43: 4098-108
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Several series of cyclin-dependent kinase inhibitors previously prepared in our laboratory were compared using 3D-QSAR (CDK1) and docking (CDK2) techniques. Evaluation of our own library of 93 purine derivatives served to establish the model which was validated by evaluation of an external library of 71 compounds. The best predictions were obtained with the CoMFA standard model (q(2) = 0.68, r(2) = 0.90) and with the CoMSIA combined steric, electrostatic, and lipophilic fields (q(2) = 0.74, r(2) = 0.90). The CDK1 3D-QSAR model was then superimposed to the ATP/CDK2 binding site, giving direct contour maps of the different fields. Although too few compounds were evaluated on CDK5 to derive a 3D-QSAR model, some interesting SARs have been deduced. Comparison of the results obtained from both methods helped with understanding the specific activity of some compounds and designing new specific CDK inhibitors.

Enke DA, Kaldis P, Solomon MJ
Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast.
J Biol Chem. 2000; 275: 33267-71
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Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 &mgr;m, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min(-)(1). The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.

Jiang GC, Yohrling GJ 4th, Schmitt JD, Vrana KE
Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling.
J Mol Biol. 2000; 302: 1005-17
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Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. The inherent instability of TPH has prevented a crystallographic structure from being resolved. For this reason, multiple sequence alignment-based molecular modeling was utilized to generate a full-length model of human TPH. Previously determined crystal coordinates of two highly homologous proteins, phenylalanine hydroxylase and tyrosine hydroxylase, were used as templates. Analysis of the model aided rational mutagenesis studies to further dissect the regulation and catalysis of TPH. Using rational site-directed mutagenesis, it was determined that Tyr235 (Y235), within the active site of TPH, appears to be involved as a tryptophan substrate orienting residue. The mutants Y235A and Y235L displayed reduced specific activity compared to wild-type TPH ( approximately 5 % residual activity). The K(m) of tryptophan for the Y235A (564 microM) and Y235L (96 microM) mutant was significantly increased compared to wild-type TPH (42 microM). In addition, kinetic analyses were performed on wild-type TPH and a deletion construct that lacks the amino terminal autoregulatory sequence (TPH NDelta15). This sequence in phenylalanine hydroxylase (residues 19 to 33) has previously been proposed to act as a steric regulator of substrate accessibility to the active site. Changes in the steady-state kinetics for tetrahydrobiopterin (BH(4)) and tryptophan for TPH NDelta15 were not observed. Finally, it was demonstrated that both Ser58 and Ser260 are substrates for Ca(2+)/calmodulin-dependent protein kinase II. Additional analysis of this model will aid in deciphering the regulation and substrate specificity of TPH, as well as providing a basis to understand as yet to be identified polymorphisms.

Prowse CN, Hagopian JC, Cobb MH, Ahn NG, Lew J
Catalytic reaction pathway for the mitogen-activated protein kinase ERK2.
Biochemistry. 2000; 39: 6258-66
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The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.

Aimes RT, Hemmer W, Taylor SS
Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: role in catalysis, P-site specificity, and interaction with inhibitors.
Biochemistry. 2000; 39: 8325-32
Display abstract
The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.

Battistutta R et al.
The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.
Eur J Biochem. 2000; 267: 5184-90
Display abstract
The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.

Mashhoon N et al.
Crystal structure of a conformation-selective casein kinase-1 inhibitor.
J Biol Chem. 2000; 275: 20052-60
Display abstract
Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

Jeffrey PD, Tong L, Pavletich NP
Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors.
Genes Dev. 2000; 14: 3115-25
Display abstract
The cyclin-dependent kinases 4 and 6 (Cdk4/6) that drive progression through the G(1) phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the INK4 family of inhibitors. INK4 proteins can bind both monomeric CDK, preventing its association with a cyclin, and also the CDK-cyclin complex, forming an inactive ternary complex. In vivo, binary INK4-Cdk4/6 complexes are more abundant than ternary INK4-Cdk4/6-cyclinD complexes, and it has been suggested that INK4 binding may lead to the eventual dissociation of the cyclin. Here we present the 2.9-A crystal structure of the inactive ternary complex between Cdk6, the INK4 inhibitor p18(INK4c), and a D-type viral cyclin. The structure reveals that p18(INK4c) inhibits the CDK-cyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18(INK4c) also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size. These observations support the model that INK4 binding weakens the cyclin's affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6.

Chaillot D, Declerck N, Niefind K, Schomburg D, Chardot T, Meunier JC
Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.
Protein Eng. 2000; 13: 291-8
Display abstract
In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Sun QY, Blumenfeld Z, Rubinstein S, Goldman S, Gonen Y, Breitbart H
Mitogen-activated protein kinase in human eggs.
Zygote. 1999; 7: 181-5
Display abstract
Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.

Zimmermann B, Chiorini JA, Ma Y, Kotin RM, Herberg FW
PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I.
J Biol Chem. 1999; 274: 5370-8
Display abstract
The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.

Graves D, Bartleson C, Biorn A, Pete M
Substrate and inhibitor recognition of protein kinases: what is known about the catalytic subunit of phosphorylase kinase?
Pharmacol Ther. 1999; 82: 143-55
Display abstract
Although much can be learned about the specificity of protein kinases from studies with peptide substrates, the question remains, how do kinases recognize their three-dimensional protein substrates? Information derived from such studies provides further understanding of substrate recognition and can facilitate the design of specific protein kinase inhibitors. Phosphorylase kinase (PhK) catalyzes the phosphorylation of phosphorylase b (phos. b) to form the active phosphorylase a. No other protein kinase can duplicate this reaction. Why? To probe this question and establish what features in the protein are important for substrate binding and product release, mutants of phos. b have been studied. This report shows how mutations change the properties of the protein substrate and the ability of these mutants to be phosphorylated by PhK and other kinases. Action of protein kinases on their substrates is often regulated by autoinhibitory segments. The C-terminus of the catalytic gamma-subunit of PhK contains two inhibitory sites overlapping two calmodulin-binding regions. These two peptide segments resemble sequences in phos. b and may explain why peptides of these regions are potent inhibitors of PhK. We will show results with peptide inhibitors, using various expressed forms of the catalytic subunit, which describe their modes of interaction and mechanisms of inhibition. Metal ions can change molecular interactions. With PhK, Mn2+ facilitates the use of GTP as a phosphoryl group donor and greatly increases phosphorylation of a tyrosine residue in angiotensin II. This implies that the spatial arrangement of specificity determinants can be manipulated so that PhK can utilize other substrates.

Taylor SS, Radzio-Andzelm E, Madhusudan, Cheng X, Ten Eyck L, Narayana N
Catalytic subunit of cyclic AMP-dependent protein kinase: structure and dynamics of the active site cleft.
Pharmacol Ther. 1999; 82: 133-41
Display abstract
The catalytic subunit of cyclic AMP-dependent protein kinase serves as a structural template for the entire family of Ser, Thr, and Tyr specific protein kinases. We review here the dynamics of the active catalytic subunit. These dynamics correlate with an opening and closing of the active site cleft, and are considered to be a requirement for catalysis. The motions, described by a set of several crystal structures, reveal a very fluid active site cleft. This active site cleft with its dynamic opening and closing is a prime target for protein kinase inhibitors.

Antonsson B, Marshall CJ, Montessuit S, Arkinstall S
An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade.
Anal Biochem. 1999; 267: 294-9
Display abstract
Mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) family are activated in response to many growth and differentiation factors as well as some oncogenes. ERK activation follows phosphorylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exemplified by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target substrates which include stathmin, a phosphoprotein regulating microtubule stability. We report here the development of a simple, 96-well plate, quantitative in vitro assay measuring purified ERK2 catalytic activation by a constitutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by 33P phosphorylation of purified biotinylated stathmin captured on streptavidin-coated scintillation proximity assay beads which eliminates the need for wash steps. The assay was optimized and the K0.5 value for ATP was found to be 0.9 microM and the Km for stathmin was determined to be 16 microM. The assay was also used to determine IC50 values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantitative measurements of MEK1-dependent ERK2 activation to be performed in a day.

Noble ME, Endicott JA
Chemical inhibitors of cyclin-dependent kinases: insights into design from X-ray crystallographic studies.
Pharmacol Ther. 1999; 82: 269-78
Display abstract
Cyclin-dependent kinases (CDKs) are a family of protein kinases that regulate progression through the eukaryotic cell cycle. Aberrant CDK activity or function is a common defect in human tumours, resulting in unrestrained cellular proliferation. X-ray crystallographic analysis of monomeric CDK2 and CDK2 complexes has revealed how phosphorylation and cyclin binding mediate enzyme activation and how this activity can be regulated by further protein association. Current research aims to improve the selectivity and/or potency of small molecule CDK inhibitors, both to develop specific probes to study the roles of the different CDK family members in coordinating cell cycle progression, and as lead molecules for the design of therapeutically useful drugs. This design process has been assisted by the availability of a number of CDK2/inhibitor structures determined using X-ray crystallography. These structures have shown that molecules related to ATP can be accommodated in the ATP-binding site in a number of orientations, utilising interactions observed between CDK2 and its natural ligand, as well as novel interactions with CDK2 residues that lie both within and outside the active site cleft. This site can also bind inhibitors that are structurally unrelated to ATP. These results suggest that it may be possible to design pharmacologically and pharmaceutically important ATP-binding site-directed ligands that act as specific and potent inhibitors of CDK activity.

Curley K, Lawrence DS
Caged regulators of signaling pathways.
Pharmacol Ther. 1999; 82: 347-54
Display abstract
Although inhibitors can implicate individual protein kinases in specific signal transduction pathways, simple reversible inhibitors are unable to define both the time of initiation and the duration of protein kinase activity. How long must a specific protein kinase remain active in a particular transduction cascade to ensure that the message results in the appropriate cellular response? What is the precise temporal assembly order of individual components of an intracellular signaling complex? What, if any, temporal crosstalk is there between individual members of bisecting signaling pathways? Caged protein kinase inhibitors, as well as caged protein kinases, should serve as useful tools for temporal studies of signal transduction. We have prepared both a caged cyclic AMP-dependent protein kinase and a caged inhibitor of this enzyme. Upon photolysis, the functional activities of both species are dramatically unleashed in vitro. We have also explored both the activation and the inhibition of the cyclic AMP-dependent protein kinase-driven signaling pathway in rat embryo fibroblasts with these caged species via microinjection, and have now demonstrated that the activity of signal transduction pathways can be controlled by light in living cells.

Zhang H, Berger JD
A novel member of the cyclin-dependent kinase family in Paramecium tetraurelia.
J Eukaryot Microbiol. 1999; 46: 482-91
Display abstract
Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.

Brown NR et al.
Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.
J Biol Chem. 1999; 274: 8746-56
Display abstract
We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Stahura FL, Xue L, Godden JW, Bajorath J
Molecular scaffold-based design and comparison of combinatorial libraries focused on the ATP-binding site of protein kinases.
J Mol Graph Model. 1999; 17: 1-9
Display abstract
Compound libraries were designed to target specifically the ATP cofactor-binding site in protein kinases by combining knowledge- and diversity-based design elements. A key aspect of the approach is the identification of molecular building blocks or scaffolds that are compatible with the binding site and therefore capture some aspects of target specificity. Scaffolds were selected on the basis of docking calculations and analysis of known inhibitors. We have generated 75 molecular scaffolds and applied different strategies to compute diverse compounds from scaffolds or, alternatively, to screen compound databases for molecules containing these scaffolds. The resulting libraries had a similar degree of molecular diversity, with at most 12% of the compounds being identical. However, their scaffold distributions differed significantly and a small number of scaffolds dominated the majority of compounds in each library.

Niefind K, Putter M, Guerra B, Issinger OG, Schomburg D
GTP plus water mimic ATP in the active site of protein kinase CK2.
Nat Struct Biol. 1999; 6: 1100-3
Display abstract
The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs that target CK2 or other kinases with this property.

Chou KC, Watenpaugh KD, Heinrikson RL
A model of the complex between cyclin-dependent kinase 5 and the activation domain of neuronal Cdk5 activator.
Biochem Biophys Res Commun. 1999; 259: 420-8
Display abstract
Tau protein kinase II (TPKII) is a heterodimer comprising a catalytic cyclin-dependent kinase subunit (Cdk5) and a regulatory protein called neuronal Cdk5 activator (Nck5a). TPKII is somewhat reminiscent, therefore, of the Cdk2-cyclin complex important in cell cycle regulation. In fact, although the amino acid sequence of Nck5a has little similarity to those of cyclins, recent experimental results obtained by site-directed mutagenesis studies have indicated that its activation domain, Nck5a*, may adopt a conformation of the cyclin-fold structure. Based on this structural inference, a 3-dimensional model of the Cdk5-Nck5a*-ATP complex was derived from the X-ray structure of Cdk2-cyclinA-ATP complex. The computed structure for TPKII is fully compatible with experimental data derived from studies of the Cdk5-Nck5a system, and also predicts which amino acid residues might be involved in formation of the Cdk5-Nck5a* interface and ATP binding pocket in TPKII. The computational structure also shows the interactive region of Nck5a* and the T-loop of Cdk5, a critical region in TPKII which functions as a gate-control-lever of the catalytic cleft. Furthermore, a physical mechanism is put forth to explain why the activation of TPKII is not dependent upon phosphorylation of the Cdk5 subunit, a puzzle long-standing in this area. These findings provide a model with which to consider design of compounds which might serve as inhibitors of TPKII.

Blachut-Okrasinska E, Lesyng B, Briggs JM, McCammon JA, Antosiewicz JM
Poisson-Boltzmann model studies of molecular electrostatic properties of the cAMP-dependent protein kinase.
Eur Biophys J. 1999; 28: 457-67
Display abstract
Protonation equilibria of residues important in the catalytic mechanism of a protein kinase were analyzed on the basis of the Poisson-Boltzmann electrostatic model along with a cluster-based treatment of the multiple titration state problem. Calculations were based upon crystallographic structures of the mammalian cAMP-dependent protein kinase, one representing the so called closed form of the enzyme and the other representing an open conformation. It was predicted that at pH 7 the preferred form of the phosphate group at the catalytically essential threonine 197 (P-Thr197) in the closed form is dianionic, whereas in the open form a monoanionic ionization state is preferred. This dianionic state of P-Thr197, in the closed form, is stabilized by interactions with ionizable residues His87, Arg165, and Lys189. Our calculations predict that the hydroxyl of the Ser residue in the peptide substrate is very difficult to ionize, both in the closed and open structures of the complex. Also, the supposed catalytic base, Asp166, does not seem to have a pK(a) appropriate to remove the hydroxyl group proton of the peptide substrate. However, when Ser of the peptide substrate is forced to remain ionized, the predicted pK(a) of Asp166 increases strongly, which suggests that the Asp residue is a likely candidate to attract the proton if the Ser residue becomes deprotonated, possibly during some structural change preceding formation of the transition state. Finally, in accord with suggestions made on the basis of the pH-dependence of kinase kinetics, our calculations predict that Glu230 and His87 are the residues responsible for the molecular pK(a) values of 6.2 and 8.5, observed in the experiment.

Steinbacher S et al.
The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.
EMBO J. 1999; 18: 2923-9
Display abstract
Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

Hauer JA, Barthe P, Taylor SS, Parello J, Padilla A
Two well-defined motifs in the cAMP-dependent protein kinase inhibitor (PKIalpha) correlate with inhibitory and nuclear export function.
Protein Sci. 1999; 8: 545-53
Display abstract
The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Kakkar R, Sharma RK
Assay and purification of calmodulin-dependent protein kinase.
Methods Mol Biol. 1999; 114: 325-41

Herberg FW, Doyle ML, Cox S, Taylor SS
Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase.
Biochemistry. 1999; 38: 6352-60
Display abstract
The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more stable by several criteria when it is part of a holoenzyme complex. By measuring the thermal stability of the free C subunit in the presence and absence of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attributed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cations as well as ATP and ADP have no effect by themselves (2). The enhanced stability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibitor/C subunit complex, metal/ATP is also required for enhanced stability; neither the RI or RII subunits nor PKI alone stabilize the C subunit significantly (4). For high thermal stability, the occupation of the second, low-affinity metal-binding site is necessary (5). From these results, we concluded that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phosphates are present, divalent metals are required for positioning these phosphates, and two metals are required to achieve thermostability comparable to adenosine alone. The complex containing two metals is the most stable. A comparison of several conformations of the C subunit derived from different crystal structures is given attributing open and closed forms of the C subunit to less and more thermostable enzymes, respectively.

Hunenberger PH, Helms V, Narayana N, Taylor SS, McCammon JA
Determinants of ligand binding to cAMP-dependent protein kinase.
Biochemistry. 1999; 38: 2358-66
Display abstract
Protein kinases are essential for the regulation of cellular growth and metabolism. Since their dysfunction leads to debilitating diseases, they represent key targets for pharmaceutical research. The rational design of kinase inhibitors requires an understanding of the determinants of ligand binding to these proteins. In the present study, a theoretical model based on continuum electrostatics and a surface-area-dependent nonpolar term is used to calculate binding affinities of balanol derivatives, H-series inhibitors, and ATP analogues toward the catalytic subunit of cAMP-dependent protein kinase (cAPK or protein kinase A). The calculations reproduce most of the experimental trends and provide insight into the driving forces responsible for binding. Nonpolar interactions are found to govern protein-ligand affinity. Hydrogen bonds represent a negligible contribution, because hydrogen bond formation in the complex requires the desolvation of the interacting partners. However, the binding affinity is decreased if hydrogen-bonding groups of the ligand remain unsatisfied in the complex. The disposition of hydrogen-bonding groups in the ligand is therefore crucial for binding specificity. These observations should be valuable guides in the design of potent and specific kinase inhibitors.

Narayana N et al.
Crystal structure of the potent natural product inhibitor balanol in complex with the catalytic subunit of cAMP-dependent protein kinase.
Biochemistry. 1999; 38: 2367-76
Display abstract
Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

Schulze-Gahmen U, Jung JU, Kim SH
Crystal structure of a viral cyclin, a positive regulator of cyclin-dependent kinase 6.
Structure Fold Des. 1999; 7: 245-54
Display abstract
BACKGROUND: Cyclin-dependent kinases (CDKs) have a central role in cell-cycle control and are activated by complex formation with positive regulatory proteins called cyclins and by phosphorylation. The overexpression and mutation of cyclins and CDKs has been associated with tumorigenesis and oncogenesis. A virus-encoded cyclin (v-cyclin) from herpesvirus saimiri has been shown to exhibit highest sequence homology to type D cyclins and specifically activates CDK6 of host cells to a very high degree. RESULTS: We have determined the first X-ray structure of a v-cyclin to 3.0 A resolution. The structure of the core domains is very similar to those of cyclin A and cyclin H from human cells. To understand the structural basis for the v-cyclin specificity for CDK6 and the insensitivity of the complex to inhibitors of the p21 and INK4 families, a v-cyclin-CDK2 model was built on the basis of the known structures of human cyclin A in complex with CDK2 and the CDK inhibitor p27(Kip1). CONCLUSIONS: Although many critical interactions between cyclin A and CDK2 would be conserved in a v-cyclin-CDK2 complex, some appear sterically or electrostatically unfavorable due to shifts in the backbone conformation or sidechain differences and may contribute to v-cyclin selectivity for CDK6. The insensitivity of v-cyclin-CDK6 complexes to inhibitors of the p21 family is probably due to structural changes in v-cyclin that lead to a flatter surface area offering fewer potential contacts with the protein inhibitor. In addition, sequence changes in v-cyclin eliminate hydrogen-bonding partners for atoms of the p27(Kip1) inhibitor. This structure provides the first model for interactions between v-cyclins and host cell-cycle proteins; these interactions may be important for virus survival as well as oncogenic transformation of host cells.

Sharma P, Steinbach PJ, Sharma M, Amin ND, Barchi JJ Jr, Pant HC
Identification of substrate binding site of cyclin-dependent kinase 5.
J Biol Chem. 1999; 274: 9600-6
Display abstract
Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.

Endicott JA, Noble ME, Tucker JA
Cyclin-dependent kinases: inhibition and substrate recognition.
Curr Opin Struct Biol. 1999; 9: 738-44
Display abstract
Four unresolved issues of cyclin-dependent kinase (CDK) regulation have been addressed by structural studies this year - the mechanism of CDK inhibition by members of the INK4 family of CDK inhibitors, consensus substrate sequence recognition by CDKs, the role of the cyclin subunit in substrate recognition and the structural mechanism underlying CDK inhibition by phosphorylation.

Yang E, Schulman H
Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II.
J Biol Chem. 1999; 274: 26199-208
Display abstract
Regulation of Ca(2+)/calmodulin-dependent protein kinase II is likely based on an auto-inhibitory mechanism in which a segment of the kinase occupies the catalytic site in the absence of calmodulin. We analyze potential auto-inhibitory associations by employing charge reversal and hydrophobic-to-charged residue mutagenesis. We identify interacting amino acid pairs by using double mutants to test which modification in the catalytic domain complements a given change in the auto-inhibitory domain. Our studies identify the core pseudosubstrate sequence (residues 297-300) but reveal that distinct sequences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn-294 in the auto-inhibitory domain or their interacting partners in the catalytic domain produces an enhanced affinity for calmodulin or generates a constitutively active enzyme. A structural model of Ca(2+)/calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why calmodulin must bind to the auto-inhibitory domain in order for Thr-286 in that domain to be phosphorylated and why introduction of phospho-Thr-286 produces the important Ca(2+)-independent state of the enzyme.

Soderling TR
The Ca-calmodulin-dependent protein kinase cascade.
Trends Biochem Sci. 1999; 24: 232-6
Display abstract
The Ca2+-calmodulin-dependent protein kinase (CaM kinase) cascade includes three kinases: CaM-kinase kinase (CaMKK); and the CaM kinases CaMKI and CaMKIV, which are phosphorylated and activated by CaMKK. Members of this cascade respond to elevation of intracellular Ca2+ levels and are particularly abundant in brain and in T cells. CaMKK and CaMKIV localize both to the nucleus and to the cytoplasm, whereas CaMKI is only cytosolic. Nuclear CaMKIV regulates transcription through phosphorylation of several transcription factors, including CREB. In the cytoplasm, there is extensive cross-talk between CaMKK, CaMKIV and other signaling cascades, including those that involve the cAMP-dependent kinase (PKA), MAP kinases and protein kinase B (PKB; also known as Akt). Activation of PKB by CaMKK appears to be important in protection of neurons from programmed cell death during development.

Tsigelny I, Greenberg JP, Cox S, Nichols WL, Taylor SS, Ten Eyck LF
600 ps molecular dynamics reveals stable substructures and flexible hinge points in cAMP dependent protein kinase.
Biopolymers. 1999; 50: 513-24
Display abstract
Molecular dynamics simulations of the catalytic subunit of cAMP dependent protein kinase (cAPK) have been performed in an aqueous environment. The relations among the protein hydrogen-bonding network, secondary structural elements, and the internal motions of rigid domains were examined. The values of fluctuations of protein dihedral angles during dynamics show quite distinct maxima in the regions of loops and minima in the regions of alpha-helices and beta-strands. Analyses of conformation snapshots throughout the run show stable subdomains and indicate that these rigid domains are constrained during the dynamics by a stable network of hydrogen bonds. The most stable subdomain during the dynamics was in the small lobe including part of the carboxy-terminal tail. The most significant flexible region was the highly conserved glycine-rich loop between beta strands 1 and 2 in the small lobe. Many of the main chain dihedral angle changes measured in a comparison of the crystallographic structures of "open" and "closed" conformations of cAPK correspond to the highly flexible residues found during dynamics.

Setyawan J et al.
Inhibition of protein kinases by balanol: specificity within the serine/threonine protein kinase subfamily.
Mol Pharmacol. 1999; 56: 370-6
Display abstract
Balanol is a potent inhibitor of cyclic AMP-dependent protein kinase and protein kinase C, acting competitively with ATP with an affinity 3000 times that of ATP. We tested the capacity of balanol to inhibit representative serine- and threonine-specific protein kinases from the protein kinase subfamily that shares a common conserved catalytic core with cyclic AMP-dependent protein kinase. Balanol's pattern of interactions indicates considerable diversity of the ATP/balanol-binding sites of protein kinases within familial groups and even among isoforms of the same kinase. We propose that balanol is a protean structure that may be modified to produce selective, high-affinity inhibitors and probes of the ATP-binding sites of serine/threonine protein kinases.

Pavletich NP
Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors.
J Mol Biol. 1999; 287: 821-8
Display abstract
The cyclin-dependent kinases (Cdks) have a central role in coordinating the eukaryotic cell division cycle, and also serve to integrate diverse growth-regulatory signals. Cdks are controlled through several different processes involving the binding of activating cyclin subunits, of inhibitory Cip or INK4 subunits, and phosphorylation. Crystallographic studies of Cdks in four different complexes, reviewed here, have revealed the mechanisms by which these regulatory processes control the Cdk switches. All of these mechanisms involve conformational changes in and around the catalytic cleft of the kinase, indicating that Cdks have evolved an intrinsic conformational flexibility. This flexibility is central to their ability to switch states in response to a diverse range of growth-regulatory signals.

Wilsbacher JL, Goldsmith EJ, Cobb MH
Phosphorylation of MAP kinases by MAP/ERK involves multiple regions of MAP kinases.
J Biol Chem. 1999; 274: 16988-94
Display abstract
Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains.

Hutter MC, Helms V
Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase.
Protein Sci. 1999; 8: 2728-33
Display abstract
The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.

Cheng X, Shaltiel S, Taylor SS
Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting.
Biochemistry. 1998; 37: 14005-13
Display abstract
Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKI resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.

Lawrence DS, Niu J
Protein kinase inhibitors: the tyrosine-specific protein kinases.
Pharmacol Ther. 1998; 77: 81-114
Display abstract
Inhibitors for tyrosine-specific protein kinases ultimately may constitute a novel family of medicinally active agents. Unfortunately, the challenges associated with the acquisition of inhibitors for these enzyme targets are unlike any that have ever been encountered in medicinal chemistry. Protein kinases pose a variety of obstacles in regard to inhibitor design, nearly all of which deal with, in one fashion or another, the issue of specificity. The protein kinase family is extraordinarily large, with estimates that the human genome codes for as many as 2000 protein kinases. Furthermore, inhibitors that are directed to the ATP-binding sites of these enzymes must contend with the presence of a large number of other ATP-utilizing proteins and, in addition, must compete with the high intracellular concentrations of ATP. Although specificity ultimately may prove to be less of a concern with peptide-based inhibitors, these agents neither are readily bioavailable nor do they bind with the requisite affinity to the protein-binding domains of protein kinases. In the face of these challenges, an enormous number of inhibitors have been synthesized and evaluated for the tyrosine-specific protein kinases. The advantages and disadvantages associated with inhibitors that are targeted to the ATP-binding site, the protein-binding site, and nonactive site regions required for appropriate subcellular localization are discussed. The handful of tyrosine-specific protein kinases that have been selected as targets to date and their roles in various disease processes are described as well.

Gangal M et al.
Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations.
Biochemistry. 1998; 37: 13728-35
Display abstract
To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.

Elion EA
Routing MAP kinase cascades.
Science. 1998; 281: 1625-6

Grant BD, Hemmer W, Tsigelny I, Adams JA, Taylor SS
Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase.
Biochemistry. 1998; 37: 7708-15
Display abstract
The conserved glycines in the glycine-rich loop (Leu-Gly50-Thr-Gly52-Ser-Phe-Gly55-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and pre-steady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s-1 in contrast to 500 s-1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy-minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.

Cheng X, Ma Y, Moore M, Hemmings BA, Taylor SS
Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase.
Proc Natl Acad Sci U S A. 1998; 95: 9849-54
Display abstract
Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.

Camps M et al.
Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase.
Science. 1998; 280: 1262-5
Display abstract
MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.

Mathews II, Erion MD, Ealick SE
Structure of human adenosine kinase at 1.5 A resolution.
Biochemistry. 1998; 37: 15607-20
Display abstract
Adenosine kinase (AK) is a key enzyme in the regulation of extracellular adenosine and intracellular adenylate levels. Inhibitors of adenosine kinase elevate adenosine to levels that activate nearby adenosine receptors and produce a wide variety of therapeutically beneficial activities. Accordingly, AK is a promising target for new analgesic, neuroprotective, and cardioprotective agents. We determined the structure of human adenosine kinase by X-ray crystallography using MAD phasing techniques and refined the structure to 1.5 A resolution. The enzyme structure consisted of one large alpha/beta domain with nine beta-strands, eight alpha-helices, and one small alpha/beta-domain with five beta-strands and two alpha-helices. The active site is formed along the edge of the beta-sheet in the large domain while the small domain acts as a lid to cover the upper face of the active site. The overall structure is similar to the recently reported structure of ribokinase from Escherichia coli [Sigrell et al. (1998) Structure 6, 183-193]. The structure of ribokinase was determined at 1.8 A resolution and represents the first structure of a new family of carbohydrate kinases. Two molecules of adenosine were present in the AK crystal structure with one adenosine molecule located in a site that matches the ribose site in ribokinase and probably represents the substrate-binding site. The second adenosine site overlaps the ADP site in ribokinase and probably represents the ATP site. A Mg2+ ion binding site is observed in a trough between the two adenosine sites. The structure of the active site is consistent with the observed substrate specificity. The active-site model suggests that Asp300 is an important catalytic residue involved in the deprotonation of the 5'-hydroxyl during the phosphate transfer.

Yang SH, Yates PR, Whitmarsh AJ, Davis RJ, Sharrocks AD
The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif.
Mol Cell Biol. 1998; 18: 710-20
Display abstract
The phosphorylation of transcription factors by mitogen-activated protein kinases (MAP) is a pivotal event in the cellular response to the activation of MAP kinase signal transduction pathways. Mitogenic and stress stimuli activate different pathways and lead to the activation of distinct groups of target proteins. Elk-1 is targeted by three distinct MAP kinase pathways. In this study, we demonstrate that the MAP kinase ERK2 is targeted to Elk-1 by a domain which is distinct from, and located N-terminally to, its phosphoacceptor motifs. Targeting via this domain is essential for the efficient and rapid phosphorylation of Elk-1 in vitro and full and rapid activation in vivo. Specific residues involved in ERK targeting have been identified. Our data indicate that the targeting of different classes of MAP kinases to their nuclear substrates may be a common mechanism to increase the specificity and efficiency of this signal transduction pathway.

Brotherton DH et al.
Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.
Nature. 1998; 395: 244-50
Display abstract
The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

Narayana N, Akamine P, Xuong NH, Taylor SS
Crystallization and preliminary X-ray analysis of the unliganded recombinant catalytic subunit of cAMP-dependent protein kinase.
Acta Crystallogr D Biol Crystallogr. 1998; 54: 1401-4
Display abstract
X-ray diffraction-quality crystals of the unliganded mouse recombinant catalytic subunit of cAMP-dependent protein kinase were grown by the hanging-drop vapour-diffusion technique using 2-methyl-2,4-pentanediol as precipitant. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 48.9, b = 147.4, c = 54.2 A, beta = 110.2 degrees. A data set to 3.0 A resolution with 92% completeness has been collected using synchrotron radiation. The unit cell contains four molecules of molecular weight 40 kDa with a corresponding volume solvent content of 45%.

Wang Y, Segall JE
The Dictyostelium MAP kinase DdERK2 functions as a cytosolic protein in complexes with its potential substrates in chemotactic signal transduction.
Biochem Biophys Res Commun. 1998; 244: 149-55
Display abstract
A polyclonal antibody against a MAP kinase (DdERK2) in Dictyostelium has been made and used to study DdERK2 activation and localization. The activation of DdERK2 by the chemoattractants cAMP and folate is rapid and transient. Its activity peaks between 15 and 60 seconds after cAMP stimulation and declines to basal levels after 5 minutes. In parallel with the DdERK2 activation is the appearance of a higher mobility band on Western blots. An antibody specific for activated MAP kinase shows that only the shifted band is tyrosine phosphorylated, suggesting that it is the active form. Both unstimulated and stimulated DdERK2 are soluble. In vitro phosphorylation with cell lysate supernatants or immunoprecipitates demonstrates the presence of several potential substrates, as identified by SDS-PAGE with mobility corresponding to molecular weights of 150, 25, and 19 kDa. Furthermore, immunoprecipitation studies suggest that these substrates are in a complex with DdERK2. These data suggest that DdERK2 works via cytoplasmic proteins to mediate signaling responses in Dictyostelium.

Xie X et al.
Crystal structure of JNK3: a kinase implicated in neuronal apoptosis.
Structure. 1998; 6: 983-91
Display abstract
BACKGROUND: The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family, and regulate signal transduction in response to environmental stress. Activation and nuclear localization of JNK3, a neuronal-specific isoform of JNK, has been associated with hypoxic and ischemic damage of CA1 neurons in the hippocampus. Knockout mice lacking JNK3 showed reduced apoptosis of hippocampal neurons and reduced seizure induced by kainic acid, a glutamate-receptor agonist. Thus, JNK3 may be important in the pathology of neurological disorders and is of significant medical interest. RESULTS: We report here the structure of unphosphorylated JNK3 in complex with adenylyl imidodiphosphate, an ATP analog. JNK3 has a typical kinase fold, with the ATP-binding site situated within a cleft between the N- and C-terminal domains. In contrast to other known MAP kinase structures, the ATP-binding site of JNK3 is well ordered; the glycine-rich nucleotide-binding sequence forms a beta-strand-turn-beta-strand structure over the nucleotide. Unphosphorylated JNK3 assumes an open conformation, in which the N- and C-terminal domains are twisted apart relative to their positions in cAMP-dependent protein kinase. The rotation leads to the misalignment of some of the catalytic residues. The phosphorylation lip of JNK3 partially blocks the substrate-binding site. CONCLUSIONS: This is the first JNK structure to be determined, providing a unique opportunity to compare structures from the three MAP kinase subfamilies. The structure reveals atomic-level details of the shape of JNK3 and the interactions between the kinase and the nucleotide. The misalignment of catalytic residues and occlusion of the active site by the phosphorylation lip may account for the low activity of unphosphorylated JNK3. The structure provides a framework for understanding the substrate specificity of different JNK isoforms, and should aid the design of selective JNK3 inhibitors.

Shaltiel S, Cox S, Taylor SS
Conserved water molecules contribute to the extensive network of interactions at the active site of protein kinase A.
Proc Natl Acad Sci U S A. 1998; 95: 484-91
Display abstract
Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. Here we summarize evidence to show that (i) the active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core; (ii) the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis; and (iii) in addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal "tail."

Pulido R, Zuniga A, Ullrich A
PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif.
EMBO J. 1998; 17: 7337-50
Display abstract
Protein kinases and phosphatases regulate the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by controlling the phosphorylation of specific residues. We report the physical and functional association of ERK1/2 with the PTP-SL and STEP protein tyrosine phosphatases (PTPs). Upon binding, the N-terminal domains of PTP-SL and STEP were phosphorylated by ERK1/2, whereas these PTPs dephosphorylated the regulatory phosphotyrosine residues of ERK1/2 and inactivated them. A sequence of 16 amino acids in PTP-SL was identified as being critical for ERK1/2 binding and termed kinase interaction motif (KIM) (residues 224-239); it was shown to be required for phosphorylation of PTP-SL by ERK1/2 at Thr253. Co-expression of ERK2 with catalytically active PTP-SL in COS-7 cells impaired the EGF-induced activation of ERK2, whereas a PTP-SL mutant, lacking PTP activity, increased the ERK2 response to EGF. This effect was dependent on the presence of the KIM on PTP-SL. Furthermore, ERK1/2 activity was downregulated in 3T3 cells stably expressing PTP-SL. Our findings demonstrate the existence of a conserved ERK1/2 interaction motif within the cytosolic non-catalytic domains of PTP-SL and STEP, which is required for the regulation of ERK1/2 activity and for phosphorylation of the PTPs by these kinases. Our findings suggest that PTP-SL and STEP act as physiological regulators of the ERK1/2 signaling pathway.

Wang Z et al.
Structural basis of inhibitor selectivity in MAP kinases.
Structure. 1998; 6: 1117-28
Display abstract
BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Schulman BA, Lindstrom DL, Harlow E
Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.
Proc Natl Acad Sci U S A. 1998; 95: 10453-8
Display abstract
An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is approximately 35A away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A-substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A's ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein-protein interaction distant from the active site is critical for phosphorylation by cdks.

Rothmann K, Schnolzer M, Radziwill G, Hildt E, Moelling K, Schaller H
Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling.
J Virol. 1998; 72: 10138-47
Display abstract
Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S-->A) or mimicking (S-->D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.

Hashimoto Y et al.
Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression.
J Biol Chem. 1998; 273: 16544-50
Display abstract
Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities. Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.

Fox T et al.
A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.
Protein Sci. 1998; 7: 2249-55
Display abstract
Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.

Khokhlatchev AV et al.
Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation.
Cell. 1998; 93: 605-15
Display abstract
The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

Godden JW, Stahura F, Bajorath J
Evaluation of docking strategies for virtual screening of compound databases: cAMP-dependent serine/threonine kinase as an example.
J Mol Graph Model. 1998; 16: 139-43
Display abstract
In an effort to establish efficient docking routines for computational screening of compound databases on protein structures, cAMP-dependent protein kinase has been selected as a test case and a variety of docking options and scoring functions were compared. These included rigid-body and flexible docking and scoring based on surface complementarity and/or force field energy. Inhibitors were removed from complex crystal structures and added to compound libraries in their binding conformations and, in addition, deliberately modified conformations. Rigid-body docking and contact scoring well reproduced two of three experimental enzyme-inhibitor complexes. Ligand docking with flexible torsional angles failed to do so but anchored search of some inhibitors converged near to experimental structures, however, only when energy scoring was applied.

Hafen E
Kinases and phosphatases--a marriage is consummated.
Science. 1998; 280: 1212-3

Johnson LN, Lowe ED, Noble ME, Owen DJ
The Eleventh Datta Lecture. The structural basis for substrate recognition and control by protein kinases.
FEBS Lett. 1998; 430: 1-11
Display abstract
Protein kinases catalyse phospho transfer reactions from ATP to serine, threonine or tyrosine residues in target substrates and provide key mechanisms for control of cellular signalling processes. The crystal structures of 12 protein kinases are now known. These include structures of kinases in the active state in ternary complexes with ATP (or analogues) and inhibitor or peptide substrates (e.g. cyclic AMP dependent protein kinase, phosphorylase kinase and insulin receptor tyrosine kinase); kinases in both active and inactive states (e.g. CDK2/cyclin A, insulin receptor tyrosine kinase and MAPK); kinases in the active state (e.g. casein kinase 1, Lck); and kinases in inactive states (e.g. twitchin kinase, calcium calmodulin kinase 1, FGF receptor kinase, c-Src and Hck). This paper summarises the detailed information obtained with active phosphorylase kinase ternary complex and reviews the results with reference to other kinase structures for insights into mechanisms for substrate recognition and control.

Takahashi M, Shimada K, Berk B C
[Fluid shear stress-mediated signal transduction in endothelial cells: temporal signaling events]
Rinsho Ketsueki. 1998; 39: 124-5

De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH
Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine.
Eur J Biochem. 1997; 243: 518-26
Display abstract
Cyclin-dependent kinases (cdk) control the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. A potent inhibitor of cdks, roscovitine [2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurin e], was identified by screening a series of C2,N6,N9-substituted adenines on purified cdc2/cyclin B. Roscovitine displays high efficiency and high selectivity (Meijer, L., Borgne, A., Mulner, O., Chong, J. P. J., Blow, J. J., Inagaki, N., Inagaki, M., Delcros, J.-G. & Moulinoux, J.-P. (1997) Eur. J. Biochem. 243, 527-536). It behaves as a competitive inhibitor for ATP binding to cdc2. We determined the crystal structure of a complex between cdk2 and roscovitine at 0.24-nm (2.4 A) resolution and refined to an Rfactor of 0.18. The purine portion of the inhibitor binds to the adenine binding pocket of cdk2. The position of the benzyl ring group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP-complex structure. Analysis of the position of this benzyl ring explains the specificity of roscovitine in inhibiting cdk2. The structure also reveals that the (R)-stereoisomer of roscovitine is bound to cdk2. The (R)-isomer is about twice as potent in inhibiting cdc2/cyclin B than the (S)-isomer. Results from structure/activity studies and from analysis of the cdk2/roscovitine complex crystal structure should allow the design of even more potent cdk inhibitors.

Zhou J, Adams JA
Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase.
Biochemistry. 1997; 36: 15733-8
Display abstract
Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.

Hemmer W, McGlone M, Tsigelny I, Taylor SS
Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.
J Biol Chem. 1997; 272: 16946-54
Display abstract
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Kawasaki H, Gotoh Y, Nishida E
[The MAP kinase cascade]
Tanpakushitsu Kakusan Koso. 1997; 42: 424-30

Okuno S, Kitani T, Fujisawa H
[CaM kinase cascade]
Tanpakushitsu Kakusan Koso. 1997; 42: 418-23

Helms V, McCammon JA
Kinase conformations: a computational study of the effect of ligand binding.
Protein Sci. 1997; 6: 2336-43
Display abstract
Protein function is often controlled by ligand-induced conformational transitions. Yet, in spite of the increasing number of three-dimensional crystal structures of proteins in different conformations, not much is known about the driving forces of these transitions. As an initial step toward exploring the conformational and energetic landscape of protein kinases by computational methods, intramolecular energies and hydration free energies were calculated for different conformations of the catalytic domain of cAMP-dependent protein kinase (cAPK) with a continuum (Poisson) model for the electrostatics. Three protein kinase crystal structures for ternary complexes of cAPK with the peptide inhibitor PKI(5-24) and ATP or AMP-PNP were modeled into idealized intermediate and open conformations. Concordant with experimental observation, we find that the binding of PKI(5-24) is more effective in stabilizing the closed and intermediate forms of cAPK than ATP. PKI(5-24) seems to drive the final closure of the active site cleft from intermediate to closed state because ATP does not distinguish between these two states. Binding of PKI(5-24) and ATP is energetically additive.

Kobe B, Heierhorst J, Kemp BE
Intrasteric regulation of protein kinases.
Adv Second Messenger Phosphoprotein Res. 1997; 31: 29-40

Miyata Y, Nishida E
[Signal transduction pathways by MAP kinase cascades]
Tanpakushitsu Kakusan Koso. 1997; 42: 1509-16

Zhou J, Adams JA
Is there a catalytic base in the active site of cAMP-dependent protein kinase?
Biochemistry. 1997; 36: 2977-84
Display abstract
The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.

Cui ZJ
[Ca2+/calmodulin-dependent protein kinase II and its functions]
Sheng Li Ke Xue Jin Zhan. 1997; 28: 61-3

Ichimura K, Mizoguchi T, Shinozaki K
[Regulation of plant MAP kinase cascade by environmental stresses and plant hormones]
Tanpakushitsu Kakusan Koso. 1997; 42: 706-16

Yonemoto W, McGlone ML, Grant B, Taylor SS
Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli.
Protein Eng. 1997; 10: 915-25
Display abstract
When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.

Tong L et al.
A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket.
Nat Struct Biol. 1997; 4: 311-6
Display abstract
The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.

Wilson KP et al.
The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase.
Chem Biol. 1997; 4: 423-31
Display abstract
BACKGROUND: The p38 mitogen-activated protein (MAP) kinase regulates signal transduction in response to environmental stress. Pyridinylimidazole compounds are specific inhibitors of p38 MAP kinase that block the production of the cytokines interleukin-1beta and tumor necrosis factor alpha, and they are effective in animal models of arthritis, bone resorption and endotoxin shock. These compounds have been useful probes for studying the physiological functions of the p38-mediated MAP kinase pathway. RESULTS: We report the crystal structure of a novel pyridinylimidazole compound complexed with p38 MAP kinase, and we demonstrate that this compound binds to the same site on the kinase as does ATP. Mutagenesis showed that a single residue difference between p38 MAP kinase and other MAP kinases is sufficient to confer selectivity among pyridinylimidazole compounds. CONCLUSIONS: Our results reveal how pyridinylimidazole compounds are potent and selective inhibitors of p38 MAP kinase but not other MAP kinases. It should now be possible to design other specific inhibitors of activated p38 MAP kinase using the structure of the nonphosphorylated enzyme.

Fadden P, Haystead TA, Lawrence JC Jr
Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes.
J Biol Chem. 1997; 272: 10240-7
Display abstract
Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

Gibson RM, Taylor SS
Dissecting the cooperative reassociation of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Role of Trp-196 in the catalytic subunit.
J Biol Chem. 1997; 272: 31998-2005
Display abstract
The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.

Lew J, Taylor SS, Adams JA
Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy.
Biochemistry. 1997; 36: 6717-24
Display abstract
The kinetics of nucleotide binding and phosphoryl group transfer were measured in the catalytic subunit of cAMP-dependent protein kinase using stopped-flow fluorescence spectroscopy and an acrylodan-labeled derivative of this enzyme, which we have previously shown to have kinetic properties similar to those for the wild-type enzyme (Lew et al., 1996). The fluorescence emission spectrum of this enzyme is quenched differentially by ATP and ADP so that both the binding of ligands and phosphoryl group transfer at the active site can be monitored selectively. The association and dissociation rate constants for both nucleotides were measured using two methods: relaxation and competition binding. The ratio of the observed dissociation and association rate constants by both methods are consistent with Kd measurements (25 microM) determined by equilibrium fluorescence quenching. The dissociation rate constant for ADP (100 s(-1)) is approximately 2.5-fold larger than k(cat) (39 s(-1)). A full viscosity effect was measured for k(cat), suggesting that a diffusive step or steps limit maximum turnover. Pre-steady-state kinetic transients are biphasic and were fitted to observed rate constants of 500 s(-1) and 60 s(-1) at 500 microM Kemptide (LRRASLG). Metal substitution studies (Mg2+ vs Mn2+) indicate that this first phase represents the phosphoryl group transfer step. Phosphopeptide release is faster than this second phase since the substrate is in rapid exchange with the enzyme and phosphorylation reduces the affinity of the peptide. The inability to assign this second phase to the chemical event or to product release implies that it reflects a viscosity-sensitive, protein conformational change that occurs after phosphoryl group transfer and prior to product release. Two conformational steps were detected in the binding of both ATP and ADP by relaxation methods that may be related to this second pre-steady-state kinetic phase. We suggest that this additional step in the kinetic mechanism may also occur in the wild-type enzyme and represents a large structural change in the enzyme during normal catalytic cycling.

Lowe ED, Noble ME, Skamnaki VT, Oikonomakos NG, Owen DJ, Johnson LN
The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.
EMBO J. 1997; 16: 6646-58
Display abstract
The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.

Newlon MG, Roy M, Hausken ZE, Scott JD, Jennings PA
The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical.
J Biol Chem. 1997; 272: 23637-44
Display abstract
Subcellular localization of the type II cAMP-dependent protein kinase is controlled by interaction of the regulatory subunit with A-Kinase Anchoring Proteins (AKAPs). This contribution examines the solution structure of a 44-residue region that is sufficient for high affinity binding to AKAPs. The N-terminal dimerization domain of the type IIalpha regulatory subunit of cAMP-dependent protein kinase was expressed to high levels on minimal media and uniformly isotopically enriched with 15N and 13C nuclei. Sequence-specific backbone and side chain resonance assignments have been made for greater than 95% of the amino acids in the free dimerization domain using high resolution multidimensional heteronuclear NMR techniques. Contrary to the results from secondary structure prediction algorithms, our analysis indicates that the domain is highly helical with a single 3-5-residue sequence involved in a beta-strand. The assignments and secondary structure analysis provide the basis for analyzing the structure and dynamics of the dimerization domain both free and complexed with specific anchoring proteins.

Jiang Y et al.
Structure-function studies of p38 mitogen-activated protein kinase. Loop 12 influences substrate specificity and autophosphorylation, but not upstream kinase selection.
J Biol Chem. 1997; 272: 11096-102
Display abstract
Several mitogen-activated protein kinase (MAPK) cascades have been identified in eukaryotic cells. The activation of MAPKs is carried out by distinct MAPK kinases (MEKs or MKKs), and individual MAPKs have different substrate preferences. Here we have examined how amino acid sequences encompassing the dual phosphorylation motif located in the loop 12 linker (L12) between kinase subdomains VII and VIII and the length and amino acid sequence of L12 influence autophosphorylation, substrate specificity, and upstream kinase selectivity for the MAPK p38. Conversion of L12 of p38 to an "ERK-like" structure was accomplished in several ways: (i) by replacing glycine with glutamate in the dual phosphorylation site, (ii) by placing a six-amino acid sequence present in L12 of ERK (but absent in p38) into p38, and (iii) by mutations of amino acid residues in loop 12. Two predominant effects were noted: (i) the Xaa residue in the dual phosphorylation motif Thr-Xaa-Tyr as well as the length of L12 influence p38 substrate specificity, and (ii) the length of L12 plays a major role in controlling autophosphorylation. In contrast, these modifications do not result in any change in the selection of p38 by individual MAPK kinases.

Narayana N, Cox S, Nguyen-huu X, Ten Eyck LF, Taylor SS
A binary complex of the catalytic subunit of cAMP-dependent protein kinase and adenosine further defines conformational flexibility.
Structure. 1997; 5: 921-35
Display abstract
BACKGROUND: cAMP-dependent protein kinase (cAPK), a ubiquitous protein in eukaryotic cells, is one of the simplest members of the protein kinase family. It was the first protein kinase to be crystallized and continues to serve as a biochemical and structural prototype for this family of enzymes. To further understand the conformational changes that occur in different liganded and unliganded states of cAPK, the catalytic subunit of cAPK was crystallized in the absence of peptide inhibitor. RESULTS: The crystal structure of the catalytic subunit of mouse recombinant cAPK (rC) complexed with adenosine was solved at 2.6 A resolution and refined to a crystallographic R factor of 21.9% with good stereochemical parameters. This is the first structure of the rC subunit that lacks a bound inhibitor or substrate peptide. The structure was solved by molecular replacement and comprises two lobes (large and small) which contain a number of conserved loops. CONCLUSIONS: The binary complex of rC and adenosine adopts an 'intermediate' conformation relative to the previously described 'closed' and 'open' conformations of other rC complexes. Based on a comparison of these structures, the induced fit that is necessary for catalysis and closing of the active-site cleft appears to be confined to the small lobe, as in the absence of the peptide the conformation of the large lobe, including the peptide-docking surface, does not change. Three specific components contribute to the closing of the cleft: rotation of the small lobe; movement of the C-terminal tail; and closing of the so-called glycine-rich loop. There is no induced fit in the large lobe to accommodate the peptide and the closing of the cleft. A portion of the C-terminal tail, residues 315-334, serves as a gate for the entry or exit of the nucleotide into the hydrophobic active-site cleft.

Sun G, Ke S, Budde RJ
Csk phosphorylation and inactivation in vitro by the cAMP-dependent protein kinase.
Arch Biochem Biophys. 1997; 343: 194-200
Display abstract
Csk is a protein tyrosine kinase that phosphorylates other protein tyrosine kinases of the Src family and down-regulates their activities. It is not known how Csk is regulated. We investigated the possibility that Csk is regulated through phosphorylation by examining if Csk can serve as an in vitro substrate for a panel of protein kinases. We found that Csk was phosphorylated by the cAMP-dependent protein kinase (PKA), but not by protein kinase C, Src, or the fibroblast growth factor receptor kinase. Csk phosphorylation in vitro by PKA is on a serine residue(s) and can reach a stoichiometry of approximately 0.6 mol phosphate per mole of enzyme. Furthermore, incubation with PKA in the presence of ATP and magnesium ion results in a time-dependent decrease in Csk kinase activity. A six-fold decrease in Csk activity (expressed as Vmax/Km ratio) was achieved due to a threefold increase in its Km and a twofold decrease in its Vmax value within 1 h of incubation with the catalytic subunit of PKA and ATP-Mg. Both phosphorylation and inactivation by PKA were blocked by a PKA-specific inhibitor. Csk mutants with a deleted SH2 or SH3 domain retained their ability to be phosphorylated and inactivated by PKA, indicating that the phosphorylation site is located within the catalytic domain. These studies suggest that the cAMP-dependent protein kinase can regulate Csk activity.

Saba-el-Leil MK, Malo D, Meloche S
Chromosomal localization of the mouse genes encoding the ERK1 and ERK2 isoforms of MAP kinases.
Mamm Genome. 1997; 8: 141-2

Canagarajah BJ, Khokhlatchev A, Cobb MH, Goldsmith EJ
Activation mechanism of the MAP kinase ERK2 by dual phosphorylation.
Cell. 1997; 90: 859-69
Display abstract
The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.

Kim SJ, Kim KH
Insulin rapidly stimulates ERK2 in the membrane of osteoblast-like UMR-106 cell.
Biochem Mol Biol Int. 1997; 43: 1023-31
Display abstract
We investigated subcellular distribution of ERK2 in osteoblast-like UMR-106 cell and explored to determine if its activities are regulated by insulin. 23%, 34% and 43% of total ERK2 were distributed in membrane, cytosol and nucleus, respectively. Insulin caused 40% increase of ERK2 content in membrane in 10 min whereas it induced approximately 50% decrease of ERK2 in cytosol in 10 min. In terms of kinase activity, insulin stimulated phosphorylation of the membrane-associated ERK2 by 2-fold and 1.8-fold in 1 min and 10 min and cytosolic ERK2 by 2.7-fold and 2.3-fold in 1 min and 10 min, respectively. In contrast, the phosphorylation of nuclear ERK2 was stimulated by insulin in time-dependent manner with maximal (3-fold) activity observed at 30 min. Insulin also increased the content of MEK2 in membrane by 2.2- to 2.6-fold in 10 min. MEK2 translocated into membrane in response to insulin may play a role in the activation of the membrane-associated ERK2 via phosphorylation.

Toledo LM, Lydon NB
Structures of staurosporine bound to CDK2 and cAPK--new tools for structure-based design of protein kinase inhibitors.
Structure. 1997; 5: 1551-6
Display abstract
Protein kinases are involved in a wide variety of signal transduction processes; their deregulation has been implicated in a number of human diseases, including cancer and disorders of the immune system. The recently determined structures of two protein kinases, cAPK and CDK2, in complex with an inhibitor, staurosporine, provide a detailed account of inhibitor-kinase interactions and inhibitor selectivity.

Hemmer W, McGlone M, Taylor SS
Recombinant strategies for rapid purification of catalytic subunits of cAMP-dependent protein kinase.
Anal Biochem. 1997; 245: 115-22
Display abstract
Knowledge of the crystal structure of the catalytic subunit (C) of cAMP-dependent protein kinase provided for the first time a molecular basis for probing function by site-directed mutagenesis. The purification of mutant C-subunits, however, presented new and unanticipated challenges due to instability, insolubility, and underphosphorylation of the altered proteins. To overcome these barriers, a rapid and efficient method for purifying recombinantly expressed C-subunits was developed. Purification to near homogeneity is achieved in less than 5 h. The procedure is based on colysis of bacteria that overexpress the C-subunit with bacteria that overexpress a poly-His-tagged mutant of the type II regulatory subunit H6RII (R213K). This mutant R-subunit with an altered cAMP binding site A forms holoenzyme rapidly in bacterial extracts, and the Ka (cAMP) for the resulting holoenzyme, 27-37 microM, is nearly 50-fold increased compared to holoenzyme formed with wild-type RII. Thus, after batchwise immobilizing the holoenzyme on Ni(2+)-resin, the free C-subunit can be directly eluted batchwise with high concentrations of cAMP. The method is described for the purification of wild-type C, with yields of approximately 5 mg/liter. In addition, a mutant subunit, C[G52S], which is defective in ATP binding and could not be isolated using previously described methods, was purified with equal efficiency.

Sheaff RJ
Regulation of mammalian cyclin-dependent kinase 2.
Methods Enzymol. 1997; 283: 173-93

Poon RY
Generation of phosphorylated cyclin-dependent kinase 2 and functional characterization of threonine-160-specific phosphatase KAP.
Methods Enzymol. 1997; 283: 283-92

Krady MM, Freyermuth S, Rogue P, Malviya AN
Pervanadate elicits proliferation and mediates activation of mitogen-activated protein (MAP) kinase in the nucleus.
FEBS Lett. 1997; 412: 420-4
Display abstract
There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen-activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.

Narayana N, Cox S, Shaltiel S, Taylor SS, Xuong N
Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.
Biochemistry. 1997; 36: 4438-48
Display abstract
The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Wang Z, Harkins PC, Ulevitch RJ, Han J, Cobb MH, Goldsmith EJ
The structure of mitogen-activated protein kinase p38 at 2.1-A resolution.
Proc Natl Acad Sci U S A. 1997; 94: 2327-32
Display abstract
The structure of mitogen-activated protein (MAP) kinase p38 has been solved at 2.1-A to an R factor of 21.0%, making p38 the second low activity MAP kinase solved to date. Although p38 is topologically similar to the MAP kinase ERK2, the phosphorylation Lip (a regulatory loop near the active site) adopts a different fold in p38. The peptide substrate binding site and the ATP binding site are also different from those of ERK2. The results explain why MAP kinases are specific for different activating enzymes, substrates, and inhibitors. A model presented for substrate and activator interactions has implications for the evolution of protein kinase cascades.

Prade L, Engh RA, Girod A, Kinzel V, Huber R, Bossemeyer D
Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.
Structure. 1997; 5: 1627-37
Display abstract
BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Padilla A, Hauer JA, Tsigelny I, Parello J, Taylor SS
Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics.
J Pept Res. 1997; 49: 210-20
Display abstract
Peptides derived from the inhibitor of cAMP-dependent protein kinase. PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala20-Ser21]PKI(5-24), and a phosphorylated form of the latter [Ala20-Ser21P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala20-Ser21]PKI(5-24) and the product [Ala20-Ser21P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains.

Wilson C, Heberle-Bors E, Vicente O
A conserved motif at the carboxy terminus of MAP kinases.
J Mol Evol. 1997; 44: 683-5

Brunet A, Pouyssegur J
Mammalian MAP kinase modules: how to transduce specific signals.
Essays Biochem. 1997; 32: 1-16
Display abstract
MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events. They are used to transduce a variety of information in organisms as diverse as yeasts, worms, flies or mammals. MAPK modules integrate signals coming from membrane receptor activation and, by the ability of MAPK to translocate into the nucleus and phosphorylate nuclear targets such as transcription factors, they relay extracellular signals into a genomic response. Since several MAPK modules transducing different information are expressed in the same cell, in yeast or in mammals, the question arises as to how fidelity is maintained between the distinct MAPK modules of a single cell. Two levels of specificity have been documented: the molecular selectivity of each enzyme for its substrate, which is particularly evident for the MKK-MAPK couple, permits specificity within one particular module; exogenous proteins, such as the yeast Ste5 protein, may serve as 'chaperone' proteins to tether all the members of a module and restrict signal transduction to this module. In mammalian cells, the MAPK modules are not strictly independent and one pathway may interfere with another. It remains to be determined whether this interference is of physiological relevance.

Lew J, Coruh N, Tsigelny I, Garrod S, Taylor SS
Synergistic binding of nucleotides and inhibitors to cAMP-dependent protein kinase examined by acrylodan fluorescence spectroscopy.
J Biol Chem. 1997; 272: 1507-13
Display abstract
We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.

Ginalski K, Lesyng B, Sowadski J, Wojciechowski M
Modelling of active forms of protein kinases: p38--a case study.
Acta Biochim Pol. 1997; 44: 557-64
Display abstract
An active form of p38 protein kinase, belonging to the mitogen-activated protein kinases subfamily, has been designed based on crystallographically known structures of two other kinases, an active form of protein kinase A (PKA) and an inactive form of extracellular signal-regulated kinase 2 (ERK2). The modelling procedure is described. Its general scheme can also be applied to other kinases. The structure of the active forms of p38 and PKA is very similar in the region which binds the substrate. The ATP-binding mode is very similar in the active forms of all the three studied kinases. Models of the active forms allow for further studies on transphosphorylation processes at the molecular level, and modelling of inhibitors competitive with ATP and/or substrates.

Taylor SS, Radzio-Andzelm E
Protein kinase inhibition: natural and synthetic variations on a theme.
Curr Opin Chem Biol. 1997; 1: 219-26
Display abstract
How a protein kinase is turned off is as critical for its physiological function as is its catalytic activity. Examination of solved crystal structures representing different protein kinase subfamilies reveals a variety of strategies that are utilized by nature to lock protein kinases into inactive conformations. Pseudosubstrate and adenine mimetic mechanisms as well as complementarity to surfaces other than the active site are effective. Although most synthetic or natural product inhibitors target the active site, specifically the ATP binding site, a remarkably high degree of specificity can be achieved which is due to the extended surface of the protein that these inhibitors occupy. Although targeting of the ATP binding site is proving to be very successful, there is also wide latitude for designing inhibitors that target other surfaces of the kinases.

Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R
Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2.
Nature. 1996; 380: 548-50
Display abstract
A network of interacting proteins controls the activity of cyclin-dependent kinase 2 (Cdk2) (refs 1,2) and governs the entry of higher eukaryotic cells into S phase. Analysis of this and other genetic regulatory networks would be facilitated by intracellular reagents that recognize specific targets and inhibit specific network connections. We report here the expression of a combinatorial library of constrained 20-residue peptides displayed by the active-site loop of Escherichia coli thioredoxin, and the use of a two-hybrid system to select those that bind human Cdk2. These peptide aptamers were designed to mimic the recognition function of the complementarity-determining regions of immunoglobulins. The aptamers recognized different epitopes on the Cdk2 surface with equilibrium dissociation constant in the nanomolar range; those tested inhibited Cdk2 activity. Our results show that peptide aptamers bear some analogies with monoclonal antibodies, with the advantages that they are isolated together with their coding genes, that their small size should allow their structures to be solved, and that they are designated to function inside cells.

Earnest S, Khokhlatchev A, Albanesi JP, Barylko B
Phosphorylation of dynamin by ERK2 inhibits the dynamin-microtubule interaction.
FEBS Lett. 1996; 396: 62-6
Display abstract
In the present study we show that purified bovine brain dynamin can be phosphorylated by MAP kinase, ERK2, with a stoichiometry of 1 mol phosphate/mol dynamin. The phosphorylated serine residue is located within the C-terminal 10 kDa of dynamin. Dynamin I phosphorylated by ERK2 can be specifically dephosphorylated by calcineurin but not by protein phosphatase 2A (PP2A). Phosphorylation of dynamin by ERK2 weakens the binding of dynamin to microtubules and inhibits dynamin's microtubule-activated GTPase activity. Stimulation of GTPase activity by either Grb2 or phospholipids was not affected by ERK2 phosphorylation, suggesting that the binding sites for Grb2 and phospholipids do not overlap with that for microtubules.

Songyang Z et al.
A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.
Mol Cell Biol. 1996; 16: 6486-93
Display abstract
We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Boisvert DC, Wang J, Otwinowski Z, Horwich AL, Sigler PB
The 2.4 A crystal structure of the bacterial chaperonin GroEL complexed with ATP gamma S.
Nat Struct Biol. 1996; 3: 170-7
Display abstract
GroEL is a bacterial chaperonin of 14 identical subunits required to help fold newly synthesized proteins. The crystal structure of GroEL with ATP gamma S bound to each subunit shows that ATP binds to a novel pocket, whose primary sequence is highly conserved among chaperonins. Interaction of Mg2+ and ATP involves phosphate oxygens of the alpha-, beta- and gamma-phosphates, which is unique for known structures of nucleotide-binding proteins. Although bound ATP induces modest conformational shifts in the equatorial domain, the stereochemistry that functionally coordinates GroEL's affinity for nucleotides, polypeptide, and GroES remains uncertain.

Gamm DM, Baude EJ, Uhler MD
The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo.
J Biol Chem. 1996; 271: 15736-42
Display abstract
Two isoforms of the catalytic subunit of cAMP-dependent protein kinase, Calpha and Cbeta1, are known to be widely expressed in mammals. Although much is known about the structure and function of Calpha, few studies have addressed the possibility of a distinct role for the Cbeta proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. Cbeta1 demonstrated higher Km values for some peptide substrates than did Calpha, but Cbeta1 was insensitive to substrate inhibition, a phenomenon that was observed with Calpha at substrate concentrations above 100 microM. Calpha and Cbeta1 displayed distinct IC50 values for the alpha and beta isoforms of the protein kinase inhibitor, protein kinase inhibitorpeptide, and the type IIalpha regulatory subunit (RIIalpha). Of particular interest, purified type II holoenzyme containing Cbeta1 exhibited a 5-fold lower Ka value for cAMP (13 nM) than did type II holoenzyme containing Calpha (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RIIalpha2Cbeta12 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RIIalpha2Calpha2 holoenzyme. These results provide evidence that type II holoenzyme formed with Cbeta1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.

Kim SH, Schulze-Gahmen U, Brandsen J, de Azevedo Junior WF
Structural basis for chemical inhibition of CDK2.
Prog Cell Cycle Res. 1996; 2: 137-45
Display abstract
The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for discovering small inhibitory molecules that can modify the degree of cell proliferation. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitors. In this article five structures of human CDK2 are summarised: apoprotein, ATP complex, olomoucine complex, isopentenyladenine complex, and des-chloro-flavopiridol complex.

Xu RM, Carmel G, Kuret J, Cheng X
Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors.
Proc Natl Acad Sci U S A. 1996; 93: 6308-13
Display abstract
A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases. One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources. Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution. The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases. Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring. We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8. Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents.

Okuno S, Fujisawa H
[Ca2+/calmodulin dependent protein kinase cascade]
Seikagaku. 1996; 68: 380-5

Yan X, Corbin JD, Francis SH, Lawrence DS
Precision targeting of protein kinases. An affinity label that inactivates the cGMP- but not the cAMP-dependent protein kinase.
J Biol Chem. 1996; 271: 1845-8
Display abstract
Although the cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) usually participate in unrelated biological processes, their enzymological properties are decidedly similar. Based upon the multitude of comparative studies conducted to date, it appears that these two enzymes exhibit very similar peptide substrate specificities. Furthermore, most inhibitors that have been reported for PKG serve in a nearly equal capacity for PKA. Consequently, the task of distinguishing between these enzymes, especially under in vivo conditions, has proved to be daunting. However, we have recently found that PKA will only phosphorylate non-amino acid residues whose alpha-configuration corresponds to that found in L-amino acids, whereas PKG will catalyze the phosphorylation of residues corresponding to both L- and D-amino acids (Wood, J., Mendelow, M., Yan, X., Corbin, J.D., Francis, S.H., and Lawrence, D.S. (1996) J. Biol. Chem. 271, 174-179). Based on these results, we have designed a potent affinity label for PKG (KI = 21.1 +/- 4.7 microM), that has no measurable activity toward PKA. This represents the first example of an peptide-based inactivator that fully distinguishes between these two closely related enzymes. These results suggest that a similar strategy may provide highly specific inactivators for other protein kinases as well.

Treptau T et al.
Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.
Biol Chem Hoppe Seyler. 1996; 377: 203-9
Display abstract
The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.

Lee SL, Steinberg RA
Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells.
J Biol Chem. 1996; 271: 16553-8
Display abstract
The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of approximately 9.2 h in unstimulated cells and approximately 4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.

Cheng M, Zhen E, Robinson MJ, Ebert D, Goldsmith E, Cobb MH
Characterization of a protein kinase that phosphorylates serine 189 of the mitogen-activated protein kinase homolog ERK3.
J Biol Chem. 1996; 271: 12057-62
Display abstract
A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser189, comparable to Thr183, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.

Li TC, Hu DY, Tang CS
[Mitogen-activated protein kinase involved in intracellular signal transduction]
Sheng Li Ke Xue Jin Zhan. 1996; 27: 258-60

Grant BD, Adams JA
Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques.
Biochemistry. 1996; 35: 2022-9
Display abstract
The phosphorylation of a peptide substrate by the catalytic subunit of cAMP-dependent protein kinase was monitored over short time periods (2-1000 ms) using a rapid quench flow mixing device and a radioactive assay. The production of phosphokemptide [LRRAS(P)LG] as a function of time is characterized by a rapid "burst" phase (250 s-1) followed by a slower, linear phase (L/[E]t = 21 s-1) at 100 microM Kemptide. The amplitude of this "burst" phase varies linearly with the enzyme concentration and represents approximately 100% of the total enzyme concentration, indicating that the "burst" phase is not due to product inhibition. The observed rate constants for the "burst" and linear phases and the "burst" amplitude vary hyperbolically with the substrate concentration. From these dependencies, a maximum "burst" rate constant of 500 +/- 60 s-1 and a Km and Kd for Kemptide of 4.9 +/- 1.4 and 200 +/- 60 microM were determined. The kcat and Km data extracted from the linear portion of the rapid quench flow transients are indistinguishable from those obtained by standard steady-state kinetic analyses using low catalytic subunit concentrations and a spectrophotometric, coupled enzyme assay. Both rate constants for the "burst" and linear phases decreased in the presence of Mn2+. The data imply that the phosphorylation of Kemptide by the catalytic subunit occurs by a mechanism in which the substrate is loosely bound, is rapidly phosphorylated at the active site, and is released at a steady-state rate that is likely controlled by the dissociation rate constant for ADP. The combined pre-steady-state kinetic data establish a comprehensive, kinetic mechanism that predicts all the steady-state kinetic and viscosometric data. This study represents the first chemical observation and characterization of phosphoryl transfer at the active site of a protein kinase and will be useful for further structure-function studies on this and other protein kinases.

Soderling TR
Structure and regulation of calcium/calmodulin-dependent protein kinases II and IV.
Biochim Biophys Acta. 1996; 1297: 131-8

Schulze-Gahmen U, De Bondt HL, Kim SH
High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: bound waters and natural ligand as guides for inhibitor design.
J Med Chem. 1996; 39: 4540-6
Display abstract
Inhibition of the cell cycle is widely considered as a new approach toward treatment for diseases caused by unregulated cell proliferation, including cancer. Since cyclin-dependent kinases (CDKs) are key enzymes of cell cycle control, they are promissing targets for the design and discovery of drugs with antiproliferative activity. The detailed structural analysis of CDK2 can provide valuable information for the design of new ligands that can bind in the ATP binding pocket and inhibit CDK2 activity. For this objective, the crystal structures of human CDK2 apoenzyme and its ATP complex were refined to 1.8 and 1.9 A, respectively. The high-resolution refinement reveals 12 ordered water molecules in the ATP binding pocket of the apoenzyme and five ordered waters in that of the ATP complex. Despite a large number of hydrogen bonds between ATP-phosphates and CDK2, binding studies of cyclic AMP-dependent protein kinase with ATP analogues show that the triphosphate moiety contributes little and the adenine ring is most important for binding affinity. Our analysis of CDK2 structural data, hydration of residues in the binding pocket of the apoenzyme, flexibility of the ligand, and structural differences between the apoenzyme and CDK2-ATP complex provide an explanation for the results of earlier binding studies with ATP analogues and a basis for future inhibitor design.

Robinson MJ et al.
Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5'-triphosphate.
Biochemistry. 1996; 35: 5641-6
Display abstract
Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

Bokemeyer D, Sorokin A, Dunn MJ
Multiple intracellular MAP kinase signaling cascades.
Kidney Int. 1996; 49: 1187-98

Sclafani RA
Cyclin dependent kinase activating kinases.
Curr Opin Cell Biol. 1996; 8: 788-94
Display abstract
The cyclin dependent kinase activating kinase (CAK) has roles in both cell cycle regulation and transcription. CAK assembly is regulated either by additional protein binding or by phosphorylation. A recent comparison of this kinase from two yeast species shows that different proteins perform distinct roles and that the most studied CAK may function mainly in transcription.

Carlson GL, Nelson DL
The 44-kDa regulatory subunit of the Paramecium cAMP-dependent protein kinase lacks a dimerization domain and may have a unique autophosphorylation site sequence.
J Eukaryot Microbiol. 1996; 43: 347-56
Display abstract
The 44-kDa regulatory subunit (R44) of one form of cAMP-dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R44 cDNA clone was 1022-bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5' and 3' untranslated sequences, respectively. A 1.1-kb mRNA was labeled on a Northern blot. The deduced R44 amino acid sequence had 31%-38% positional identity to the sequences of other cloned cAMP-dependent protein kinase regulatory subunits. R44 sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N-terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R44 clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP-dependent protein kinase. The putative autophosphorylation site of R44 contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP-dependent protein kinase regulatory subunits and many cAMP-dependent protein kinase substrates.

Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP
Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex.
Nature. 1996; 382: 325-31
Display abstract
The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.

Bourne Y et al.
Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1.
Cell. 1996; 84: 863-74
Display abstract
The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe. This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites. CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation. The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions. One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle.

Goldberg J, Nairn AC, Kuriyan J
Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I.
Cell. 1996; 84: 875-87
Display abstract
The crystal structure of calcium/calmodulin-dependent protein kinase I has been determined in the autoinhibited form. The C-terminal regulatory region of the enzyme forms a helix-loop-helix segment that extends across the two domains of the catalytic core, making multiple inhibitory interactions. Elements of the first regulatory alpha helix and the loop interfere with the binding site for peptide substrates, while the loop and the second helix interact with the ATP-binding domain to induce conformational changes that obstruct the nucleotide binding pocket. One part of the calmodulin recognition element protrudes away from the catalytic domain and is potentially available for an initial interaction with calmodulin. The structure provides a view of an intact calmodulin target and suggests that substantial structural changes will accompany kinase activation by calmodulin binding to the regulatory region.

Picciotto MR, Nastiuk KL, Nairn AC
Structure, regulation, and function of calcium/calmodulin-dependent protein kinase I.
Adv Pharmacol. 1996; 36: 251-75

Cobb MH et al.
Structural analysis of the MAP kinase ERK2 and studies of MAP kinase regulatory pathways.
Adv Pharmacol. 1996; 36: 49-65

Cohen P
Dissection of protein kinase cascades that mediate cellular response to cytokines and cellular stress.
Adv Pharmacol. 1996; 36: 15-27

Iino M, Shibano T
Substrate recognition mechanism of human beta-adrenergic receptor kinase 1 based on a three-dimensional model structure.
Drug Des Discov. 1996; 14: 145-55
Display abstract
Although its detailed substrate specificity is not precisely known, beta-adrenergic receptor kinase 1 phosphorylates beta 2-adrenergic receptors and other G protein-coupled receptors. To elucidate the ligand recognition mechanism of the enzyme and the consensus sequence required for substrates, a three-dimensional structure of the catalytic domain of the enzyme was modeled based on the X-ray crystal structure of the catalytic subunit of cyclic AMP-dependent protein kinase A. When the phosphorylation residue of the substrate was defined as the p position in the model of beta-adrenergic receptor kinase 1, the present study suggested that the consensus sequence recognized by this enzyme would consist of a basic residue at p-3 and an acidic residue at p-2.

Grant BD, Tsigelny I, Adams JA, Taylor SS
Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.
Protein Sci. 1996; 5: 1316-24
Display abstract
The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.

Longenecker KL, Roach PJ, Hurley TD
Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition.
J Mol Biol. 1996; 257: 618-31
Display abstract
The three-dimensional structure for the catalytic region of the mammalian protein kinase, casein kinase I delta (CKI delta), has been solved by X-ray crystallography to a resolution of 2.3 A. A truncation mutant of CKI delta lacking the C-terminal autoinhibitory region was expressed in Escherichia coli, purified, and crystallized. The structure was solved by molecular replacement using the crystal structure of the catalytic domain of a CKI homolog from Schizosaccharomyces pombe, Cki1. A tungstate derivative confirmed the initial molecular replacement solution and identified an anion binding site which may contribute to the unique substrate specificity of CKI. Like other protein kinases, the catalytic domain of CKI is composed of two lobes with a cleft between them for binding ATP. Comparison of the mammalian and yeast CKI structures suggests that a rotation of the N-terminal domain occurs upon ATP binding. This domain motion is similar, but not identical, to that observed in cAMP-dependent protein kinase upon binding ATP. Although Cki1 has many similarities to CKI delta over the catalytic domain, these two forms of CKI likely perform different functions in vivo. Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI delta reveals a catalytic face that is especially conserved among the subset of CKI family members associated with the regulation of DNA repair.

Tsigelny I, Grant BD, Taylor SS, Ten Eyck LF
Catalytic subunit of cAMP-dependent protein kinase: electrostatic features and peptide recognition.
Biopolymers. 1996; 39: 353-65
Display abstract
The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5-24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923-8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pKa shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pKa shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pKa shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and delta pKa have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer.

Sahara S et al.
Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit.
FEBS Lett. 1996; 384: 138-42
Display abstract
To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

Russo AA, Jeffrey PD, Pavletich NP
Structural basis of cyclin-dependent kinase activation by phosphorylation.
Nat Struct Biol. 1996; 3: 696-700
Display abstract
Cyclin-dependent kinase (CDK)-cyclin complexes require phosphorylation on the CDK subunit for full activation of their Ser/Thr protein kinase activity. The crystal structure of the phosphorylated CDK2-CyclinA-ATP gamma S complex has been determined at 2.6 A resolution. The phosphate group, which is on the regulatory T-loop of CDK2, is mostly buried, its charge being neutralized by three Arg side chains. The arginines help extend the influence of the phosphate group through a network of hydrogen bonds to both CDK2 and cyclinA. Comparison with the unphosphorylated CDK2-CyclinA complex shows that the T-loop moves by as much as 7 A, and this affects the putative substrate binding site as well as resulting in additional CDK2-CyclinA contacts. The phosphate group thus acts as a major organizing centre in the CDK2-CyclinA complex.

Werner DS, Lee TR, Lawrence DS
Is protein kinase substrate efficacy a reliable barometer for successful inhibitor design?
J Biol Chem. 1996; 271: 180-5
Display abstract
We have addressed the question of whether protein kinase substrate efficacy is a reliable barometer for successful inhibitor design by assessing the dependence of kcat and kcat/Km for eight separate alcohol-bearing residues on solvent viscosity. We have found that the Km for three structurally distinct primary alcohol-containing peptides overestimates the affinity that these species exhibit for the cAMP-dependent protein kinase. In all three cases, the rate-determining step is product release, and substrate binding is best described as rapid equilibrium. In contrast, peptides containing the following phosphorylatable residues all provide Km values that are accurate assessments of substrate affinity for the protein kinase: a secondary alcohol, a simple phenol, and a primary alcohol with a relatively long side chain. In the latter three instances, the rate-determining step is phosphoryl transfer. Finally, two aromatic alcohol-containing residues that possess lipophilic side chains exhibit Michaelis constants that underestimate enzyme affinity. These results demonstrate that while it may be tempting to employ structural elements from the most efficient substrates (e.g. primary alcohols) for inhibitor design, less effective substrates may serve as a more accurate assessment of inhibitory success.

Fotedar R et al.
p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity.
Oncogene. 1996; 12: 2155-64
Display abstract
Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21Cip1/Waf1/Sdi1 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase delta. The kinase inhibitory domain maps to the N-terminus (1-82) and contains the cdk2 binding site (28-82). We have generated a panel of deletion mutants of p21. A functional characterization of p21 mutants in the N-terminal domain reveals that cyclins bind to this domain independently of cdk2. Correlating with these results we find that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.

Ruth P, Kamm S, Nau U, Pfeifer A, Hofmann F
A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).
Biol Chem. 1996; 377: 513-20
Display abstract
Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Sowadski JM, Ellis CA, Madhusudan
Detergent binding to unmyristylated protein kinase A--structural implications for the role of myristate.
J Bioenerg Biomembr. 1996; 28: 7-12
Display abstract
Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knighton et al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlsson et al., 1993; Zheng et al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zheng et al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This "inside out" motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60v-src.

Zaliani A, Gromo G, Pinori M, Mascagni P
Rational design of a new C-myristylamido peptide exerting potent and selective PKC inhibitory activity.
Drug Des Discov. 1996; 13: 63-74
Display abstract
A 3D model of the catalytic domain of PKC was built based on the X-ray structure of the homologous PKA enzyme. The two enzymes were found to have similar general architecture although differing for the number of negatively charged clusters and their location near the phosphorylation site. These differences were consistent with the charge requirements deduced from the consensus sequence of PKC and PKA substrates. A Myristyl Binding Site (MBS) was found in the PKC model between helix C and sheets 8 and 9. The identification of this MBS allowed the rationalization of the results obtained with N-myristoylated peptide inhibitors and, above all, the design of ITF1671 (H-RFARKGALRQKN-CONH-Myr), a new C-myristylamido peptide, which exerted one of the most potent inhibitory activity against PKC and PKM known to-date.

Kim KK, Chamberlin HM, Morgan DO, Kim SH
Three-dimensional structure of human cyclin H, a positive regulator of the CDK-activating kinase.
Nat Struct Biol. 1996; 3: 849-55
Display abstract
Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at a specific threonine residue. Vertebrate CAK contains two key components: a kinase subunit with homology to its substrate CDKs and a regulatory subunit with homology to cyclins. We have determined the X-ray crystal structure of the regulatory subunit of CAK, cyclin H, at 2.6 A resolution. Cyclin H contains two alpha-helical core domains with a fold similar to that of cyclin A, a regulatory subunit of CAK substrate CDK2, and of TFIIB, a transcription factor. Outside of the core domains, the N- and C-terminal regions of the three structures are completely different. The conformational differences between cyclin H and A structures may reflect functional differences between the two cyclins.

Allende JE, Allende CC
Protein kinases. 4. Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation.
FASEB J. 1995; 9: 313-23
Display abstract
Protein kinase CK2 (also known as casein kinase II) is a ubiquitous eukaryotic ser/thr protein kinase present in the nucleus and cytoplasm. CK2 is known to phosphorylate more than 100 substrates, many of which are involved in the control of cell division and in signal transduction. The review centers on the structure and function of CK2 alpha and beta subunits and on the regulation of its activity, a topic that remains to be elucidated. An analogy is drawn between CK2 and the cyclin-dependent kinases (cdks); both types of protein kinases share many substrates and are activated by regulatory subunits.

Reuter CW, Catling AD, Weber MJ
Immune complex kinase assays for mitogen-activated protein kinase and MEK.
Methods Enzymol. 1995; 255: 245-56

Kortenjann M, Shaw PE
The growing family of MAP kinases: regulation and specificity.
Crit Rev Oncog. 1995; 6: 99-115
Display abstract
The family of MAP kinases consists of several subgroups of serine/threonine protein kinases. Together with their activating kinases, they function to regulate cellular responses to diverse extracellular signals, including osmotic stress, heat shock, proinflammatory cytokines, and mitogens. It is now clear that as in yeast, separate MAP kinase cascades exist in mammalian cells, responding selectively to different stimuli by phosphorylating cytoplasmic components and nuclear transcription factors. Down-regulation of MAP kinase pathways may occur through dephosphorylation by serine/threonine phosphatases, tyrosine phosphatases, or dual-specificity phosphatases and through feedback inhibitory mechanisms that involve the phosphorylation of upstream kinases. The functional integrity of each MAP kinase cascade is thought to be established and maintained by specific molecular interactions both between the kinases and with cytoplasmic anchors that nucleate complex formation. The recent demonstration that a series of pyridinyl-imidazole compounds can bind and inhibit certain MAP kinases suggests that other MAP kinase subgroups may also be susceptible to synthetic compounds. Drugs that selectively down-regulate MAP kinase cascades could prove to be valuable as therapeutic agents in the control of malignant disease.

Keyse SM
An emerging family of dual specificity MAP kinase phosphatases.
Biochim Biophys Acta. 1995; 1265: 152-60

Han J, Richter B, Li Z, Kravchenko V, Ulevitch RJ
Molecular cloning of human p38 MAP kinase.
Biochim Biophys Acta. 1995; 1265: 224-7
Display abstract
Mitogen-activated protein (MAP) kinases are intracellular serine/threonine kinases activated by dual phosphorylation of adjacent threonine (T) and tyrosine (Y). A diverse number of extracellular signals induce activation of MAP kinases. Here we describe the cloning of a cDNA encoding human p38 MAP kinase (p38). The amino acid sequence of human p38 is 99.4% identical to mouse p38 [Han et al. (1994) Science 265, 808-11]. Like murine p38, the dual phosphorylation site of human p38 MAP kinase is characterized by a TGY sequence. Previous studies have described activation of p38 following exposure to products of microbial pathogens, physical-chemical stimuli and cytokines. The highly conserved nature of p38 suggests the importance of its function in regulating cellular responses.

Adams JA, McGlone ML, Gibson R, Taylor SS
Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase.
Biochemistry. 1995; 34: 2447-54
Display abstract
Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Jeffrey PD et al.
Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.
Nature. 1995; 376: 313-20
Display abstract
The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

Kohn LD
Thyroglobulin--a new cyclic adenosine monophosphate-dependent protein kinase?
Endocrinology. 1995; 136: 3177-8

Brown NR et al.
The crystal structure of cyclin A.
Structure. 1995; 3: 1235-47
Display abstract
BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

Radzio-Andzelm E, Lew J, Taylor S
Bound to activate: conformational consequences of cyclin binding to CDK2.
Structure. 1995; 3: 1135-41
Display abstract
The cyclin-dependent kinases (CDKs) are among the most highly regulated enzymes in the protein-kinase family. The crystal structures of cyclin A and the CDK2-cyclin A complex spectacularly reveal the atomic basis for regulation of these enzymes and provide a template for understanding the function and regulation of other members of the CDK family.

Levine K, Cross FR
Structuring cell-cycle biology.
Structure. 1995; 3: 1131-4
Display abstract
The recent determination of the crystal structures of both cyclin A and the cyclin a-CDK2 complex provides new insight into the cyclin- dependent activation of cyclin-dependent protein kinases.

Lois AF, Cooper LT, Geng Y, Nobori T, Carson D
Expression of the p16 and p15 cyclin-dependent kinase inhibitors in lymphocyte activation and neuronal differentiation.
Cancer Res. 1995; 55: 4010-3
Display abstract
The cyclin-dependent kinase inhibitors p16INK4/MTS1 and p15INK4B/MTS2 have been mapped to a region in chromosome 9 (921) that is deleted frequently in acute lymphoblastic leukemias and malignant gliomas. To gain insight into the functions of these inhibitors in lymphocytes and neuronal cells, we studied the expression of p15 and p16 during lymphocyte mitogenesis and neuronal differentiation. Expression of p15 was extinguished during lymphocyte activation, concomitant with an increase in retinoblastoma kinase activity. The differentiation of the embryonic teratocarcinoma cell line NT2 into postmitotic neurons (hNT) was associated with enhanced expression of p15 and p16 proteins. These findings suggest that p15 and p16 play a role in maintaining cell quiescence in lymphocytes and neuronal cells, respectively. Deletions of these genes may thus promote unrestrained growth.

Lees E
Cyclin dependent kinase regulation.
Curr Opin Cell Biol. 1995; 7: 773-80
Display abstract
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and their activities are consequently tightly regulated. Recent developments in the field of CDK regulation have included the discovery and characterization of CDK inhibitors. These developments have had an impact on our understanding of how other signalling pathways may be linked to the cell cycle machinery.

Furet P et al.
Modelling study of protein kinase inhibitors: binding mode of staurosporine and origin of the selectivity of CGP 52411.
J Comput Aided Mol Des. 1995; 9: 465-72
Display abstract
A model for the binding mode of the potent protein kinase inhibitor staurosporine is proposed. Using the information provided by the crystal structure of the cyclic-AMP-dependent protein kinase, it is suggested that staurosporine, despite a seemingly unrelated chemical structure, exploits the same key hydrogen-bond interactions as ATP, the cofactor of the protein kinases, in its binding mode. The structure-activity relationship of the inhibitor and a docking analysis give strong support to this hypothesis. The selectivity of the dianilinophthalimide inhibitor CGP 52411 towards the EGF-receptor protein tyrosine kinase is rationalized on the basis of the model. It is proposed that this selectivity originates in the occupancy, by one of the anilino moieties of the inhibitor, of the region of the enzyme cleft that normally binds the ribose ring of ATP, which appears to possess a marked lipophilic character in this kinase.

Meador WE, George SE, Means AR, Quiocho FA
X-ray analysis reveals conformational adaptation of the linker in functional calmodulin mutants.
Nat Struct Biol. 1995; 2: 943-5

Cobb MH, Goldsmith EJ
How MAP kinases are regulated.
J Biol Chem. 1995; 270: 14843-6

Cox S, Taylor SS
Kinetic analysis of cAMP-dependent protein kinase: mutations at histidine 87 affect peptide binding and pH dependence.
Biochemistry. 1995; 34: 16203-9
Display abstract
Mutation of His87 in the catalytic (C-) subunit of the cAMP-dependent protein kinase (cAPK) led to changes in the kinetic properties of this enzyme. The C-subunit is a bilobal structure, with catalysis occurring in the cleft between the two lobes. His87 lies at the edge of the cleft, making an interaction with phosphothreonine, 197. This is the only direct electrostatic or hydrogen-bonding interaction between the small and large lobes. Solvent viscosity studies of the His87Ala mutant of the C-subunit (rC[H87A]) revealed that binding of two peptides, LRRASLG and LRRASLG-NH2, was impaired relative to that of the wild-type C-subunit. Consistent with this, the Ki's for two inhibitor peptides, LRRAALG and LRRAALG-NH2, were 4 and 1.4 mM, respectively, 5- and 7-fold higher than the Ki's of the respective peptides for wild-type protein. Kinetic constants for three octapeptide substrates that differed only at the P+2 position suggested a direct interaction of His87 with residues at this site. The kcat for rC[H87A] was 2-3-fold higher than kcat for the wild-type enzyme, indicating an effect of the mutation on the rate-limiting step, product release. The pH dependence of kinetic parameters for rC[H87A] was also measured. A single pKa of 6.5 was observed in kcat/Kpeptide as compared to the two pKa's of 6.5 and 8.5 for the wild-type enzyme. These changes suggest a role for His87 in substrate recognition and in stabilization of the catalytically competent conformation of the enzyme.

Lee JD, Ulevitch RJ, Han J
Primary structure of BMK1: a new mammalian map kinase.
Biochem Biophys Res Commun. 1995; 213: 715-24
Display abstract
Mitogen-activated protein (MAP) kinases comprise a family of conserved, eukaryotic enzymes that mediate responses to a wide variety of extracellular stimuli. We have identified a new human MAP kinase gene here termed BMK1. BMK1 encodes a protein of 816 amino acid residues and has at least three different forms of mRNA. BMK1 messages are abundant in heart, placenta and kidney but not detectable in liver. Although BMK1 has the dual phosphorylation site of MAP kinases characterized by the TEY sequence found in ERK1 and ERK2, it has a distinct C-terminal and loop-12 structure when compared to other mammalian MAP kinases. This suggests BMK1 may regulate signaling events distinct from those controlled by the ERK group of enzymes.

Orr JW, Newton AC
Intrapeptide regulation of protein kinase C.
J Biol Chem. 1994; 269: 8383-7
Display abstract
Protein kinase C is allosterically activated by diacylglycerol and phosphatidylserine. The enzyme is also activated by surprisingly dissimilar compounds such as short chained phosphatidylcholines and protamine sulfate. Here we show that conventional and nonconventional activators of protein kinase C beta II produce the same structural alteration. They expose Arg19 of the autoinhibitory pseudosubstrate domain to proteolysis. Molecular modeling of protein kinase C beta II's catalytic domain, based on the structure of the cAMP-dependent kinase's catalytic domain, indicates that Arg19 is shielded by a cluster of acidic residues when the pseudosubstrate occupies the substrate-binding site. Our biochemical data and structural modeling indicate a common mechanism of intrapeptide regulation of protein kinase C by all activators that involves release of the pseudosubstrate from the active site.

Traut TW
The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide-binding sites.
Eur J Biochem. 1994; 222: 9-19
Display abstract
From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTP. Binding motif Kinase-1a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP). NMP-1 and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-1 is found in almost all the kinases that phosphorylate AMP, CMP, GMP, dTMP, or UMP. NMP-3a is found in kinases for AMP, GMP, and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nucleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the gamma-phosphate, or of the adenylate to an acceptor) and also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein.

Tarkka T, Yli-Mayry N, Mannermaa RM, Oikarinen J
Nucleotide recognition by histone H1 involves specific protein structures.
Arch Biochem Biophys. 1994; 310: 291-9
Display abstract
We have reported previously that histone H1 is capable of binding nucleotides such as ATP, GTP, ADP, and GDP in a specific manner. It is demonstrated here using labeling with the uv-crosslinkable ATP analog 8-azido-[alpha-32P]ATP that this ability is a unique characteristic of H1 among the histone proteins. Phosphate analogs such as AlF-4 efficiently counteract the labeling of H1, while they do not compete for labeling of histones H2A, H2B, H3, and H4. Consistent with the assumption that this labeling is due to specific binding, nucleotides competed for the labeling of H1 in a manner similar to labeling of the catalytic subunit of cAMP-dependent protein kinase, casein kinase-II, and heat shock protein-90, all of which are ATP/GTP-binding proteins. The site of nucleotide interaction was subsequently located in a Gly-rich region of H1 which displays homology with the protein kinases, using either radioactive labeling with nucleotide analogs and endoproteinase Glu-C digestion or synthetic peptides corresponding to the putative binding site. The results imply that specific protein structures are involved in nucleotide binding to H1 and that the ability of H1 to bind nucleotides may provide a mechanism for the regulation of eukaryotic gene expression.

Nebreda AR
Inactivation of MAP kinases.
Trends Biochem Sci. 1994; 19: 1-2

Kalivas PW
Blockade of neurotensin-induced motor activity by inhibition of protein kinase.
Psychopharmacology (Berl). 1994; 114: 175-80
Display abstract
The administration of neurotensin into the ventral tegmental area stimulates dopamine neurons and locomotor activity. Furthermore, when neurotensin is microinjected daily into the ventral tegmental area the motor stimulant response increases. The role of protein kinases in the motor stimulant effect of neurotensin was evaluated by coadministration of the protein kinase inhibitors H8 and H7 into the ventral tegmental area with neurotensin. It was found that the acute motor stimulant effect of neurotensin was abolished in a dose-dependent fashion by H8 coadministration. Neurotensin-induced activity was also blocked by H7. However, acute motor stimulation following microinjection of the mu opioid, Tyr-d-Ala-Gly-MePhe-Gly(ol) or the potassium channel antagonist apamin into the ventral tegmental area was not affected by coadministration with H8. The behavioral sensitization produced by daily neurotensin microinjection into the ventral tegmental area was also prevented by the coadministration of H8. These data indicate that the motor stimulation produced by acute and repeated neurotensin microinjection into the ventral tegmental area is dependent upon activation of protein kinase(s). Furthermore, Tyr-d-Ala-Gly-MePhe-Gly(ol) and apamine elicit locomotion independently of protein kinase(s).

Robbins DJ, Zhen E, Cheng M, Xu S, Ebert D, Cobb MH
MAP kinases ERK1 and ERK2: pleiotropic enzymes in a ubiquitous signaling network.
Adv Cancer Res. 1994; 63: 93-116

Vesely J et al.
Inhibition of cyclin-dependent kinases by purine analogues.
Eur J Biochem. 1994; 224: 771-86
Display abstract
While testing purines related to the non-specific protein kinase inhibitors N6-dimethylaminopurine and N6-(delta 2-isopentenyl)adenine as potential inhibitors of the p34cdc2/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6- benzylamino-9-methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34cdk4/cyclin D1, p40cdk6/cyclin D3, cAMP-dependent and cGMP-dependent kinases, eight protein kinase C isoforms, calmodulin-dependent kinase II, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded RNA-activated protein kinase, AMP-stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase (and its starfish homologue p44mpk) are substantially inhibited by olomoucine (IC50 values are 7, 7, 7, 3 and 25 microM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC50 values greater than 1 mM and 150 microM, respectively). N6-(delta 2-Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC50 values of 50-100 microM for many kinases. The purine specificity of cyclin-dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9-substituted purines exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain-specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M-phase-promoting factor activity in metaphase-arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti-mitotic reagent that may preferentially inhibit certain steps of the cell cycle.

Wen W, Taylor SS
High affinity binding of the heat-stable protein kinase inhibitor to the catalytic subunit of cAMP-dependent protein kinase is selectively abolished by mutation of Arg133.
J Biol Chem. 1994; 269: 8423-30
Display abstract
The two classes of physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase are the regulatory subunits and the heat-stable protein kinase inhibitors (PKIs), and both share a common mechanism of inhibition. Each has a similar inhibitor site that resembles a peptide substrate, and this occupies the P-3 to P+1 portion of the peptide recognition site. However, in addition to this consensus site, each inhibitor requires a peripheral binding site to achieve high affinity binding. Arg134 and Arg133 lie on the surface of the catalytic subunit with Arg133 coming close to the amphipathic helix of PKI(5-24) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-h., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420). Replacement of Arg134 and Arg133 with Ala selectively abolishes the high affinity binding of PKI. Replacement of Arg133 alone is sufficient to give the same phenotype. In the presence of MgATP, the Kd,app, is increased from < 0.2 to 105 nM and, in the absence of ATP, the Kd is too large to be reliably measured. Based on the crystal structure, Arg133 hydrogen bonds to the P-7 backbone carbonyl of PKI(5-24). However, more importantly, it also contributes to the hydrophobicity of the P-11 binding site in the C.PKI(5-24) complex. We predict that it is the perturbation of this hydrophobic pocket that accounts for the effects of this mutation. In the absence of peptide, Arg133 may help to stabilize Glu230, a buried carboxylate that binds to the P-2 Arg in the crystal structure of C.PKI(5-24). Replacement of Arg133 and Arg134 with Ala has little effect on catalysis using a heptapeptide substrate and has no effect on the inhibition of the catalytic subunit by the regulatory subunit. The results thus demonstrate that these two inhibitor proteins that both bind to the catalytic subunit with a high affinity utilize different sites on the enzyme to achieve tight binding. The gamma isoform of the catalytic subunit is insensitive to inhibition by PKI and in this isoform Arg133 is replaced with Gln. We predict that this change accounts for the altered inhibitor properties of C gamma.

Songyang Z, Blechner S, Hoagland N, Hoekstra MF, Piwnica-Worms H, Cantley LC
Use of an oriented peptide library to determine the optimal substrates of protein kinases.
Curr Biol. 1994; 4: 973-82
Display abstract
BACKGROUND: Phosphorylation by protein kinases is an important general mechanism for controlling intracellular processes, and plays an essential part in the signal transduction pathways that regulate cell growth in response to extracellular signals. A great number of protein kinases have been discovered, and the identification of their biological targets is still a very active research area. Protein kinases must have the appropriate substrate specificity to ensure that signals are transmitted correctly. Previous studies have demonstrated the importance of primary sequences within substrate proteins in determining protein kinase specificity, but efficient ways of identifying these sequences are lacking. RESULTS: We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates. In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. The optimal substrate predicted for cAMP-dependent protein kinase (PKA) by this technique is consistent with the sequences of known PKA substrates. The optimal sequences predicted for cyclin-dependent kinases (CDKs) cyclin B-Cdc2 and cyclin A-CDK2 also agree well with sites thought to be phosphorylated in vivo by these kinases. In addition, we determined the optimal substrate for SLK1, a homologue of the STE20 protein serine kinase of hitherto unknown substrate specificity. We also discuss a model incorporating the optimal cyclin B-Cdc2 substrate into the known crystal structure of this kinase. CONCLUSIONS: Using the new technique we have developed, the sequence specificity of protein kinases can rapidly be predicted and, from this information, potential targets of the kinases can be identified.

Cobb MH, Xu S, Hepler JE, Hutchison M, Frost J, Robbins DJ
Regulation of the MAP kinase cascade.
Cell Mol Biol Res. 1994; 40: 253-6
Display abstract
The MAP kinase cascade is regulated by many hormones and growth factors and its activation leads to changes in properties of cytoplasmic, membrane-associated, and nuclear proteins. The MAP kinases themselves are activated by MEKS. MEKs lie at a point of convergence for multiple upstream signals, mediated by distinct protein kinases, Raf, MEK kinase, and Mos, all of which have MEK kinase activity. Additional inputs that stimulate the MAP kinase pathway are the activation of protein kinase C and the yeast protein kinase STE20. Mechanisms of regulation of some of the upstream components of this cascade have not yet been fully elucidated.

Marshall CJ
Signal transduction. Hot lips and phosphorylation of protein kinases.
Nature. 1994; 367: 686-686

Blumer KJ, Johnson GL
Diversity in function and regulation of MAP kinase pathways.
Trends Biochem Sci. 1994; 19: 236-40
Display abstract
Eukaryotic cells from yeast to humans use sequential protein kinase reactions to regulate complex cellular functions. Equivalent protein kinases in different pathways have significant sequence homologies; however, little crossover in phosphorylation of substrates between pathways normally occurs. Assembly of kinase complexes and discrimination of substrates provide the selectivity of sequential protein kinase pathways to regulate such diverse cellular functions as osmoregulation, cell-wall biosynthesis, growth and differentiation.

Madhusudan et al.
cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.
Protein Sci. 1994; 3: 176-87
Display abstract
The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

Cobb MH, Hepler JE, Cheng M, Robbins D
The mitogen-activated protein kinases, ERK1 and ERK2.
Semin Cancer Biol. 1994; 5: 261-8
Display abstract
The MAP kinases are ubiquitous enzymes that are activated in a complex fashion and inactivated by multiple phosphatases including a dedicated dual specificity enzyme. These kinases have a diverse array of substrates with important functions that result in their substantial regulatory impact. The ERK/MAP kinase cascade displays not only downstream but also upstream interactions as well as cross talk with other signaling pathways which fine tunes the cascade in a cell type-specific fashion. Transforming agents utilize this cascade in inducing cell proliferation.

Morgan DO, De Bondt HL
Protein kinase regulation: insights from crystal structure analysis.
Curr Opin Cell Biol. 1994; 6: 239-46
Display abstract
The crystal structures of three protein kinases in various states of activity have recently been determined. Analysis of these structures is providing unprecedented insight into the precise atomic movements underlying protein kinase regulation.

Han J, Lee JD, Bibbs L, Ulevitch RJ
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.
Science. 1994; 265: 808-11
Display abstract
Mammalian cells respond to endotoxic lipopolysaccharide (LPS) by activation of protein kinase cascades that lead to new gene expression. A protein kinase, p38, that was tyrosine phosphorylated in response to LPS, was cloned. The p38 enzyme and the product of the Saccharomyces cerevisiae HOG1 gene, which are both members of the mitogen-activated protein (MAP) kinase family, have sequences at and adjacent to critical phosphorylation sites that distinguish these proteins from most other MAP kinase family members. Both HOG1 and p38 are tyrosine phosphorylated after extracellular changes in osmolarity. These findings link a signaling pathway in mammalian cells with a pathway in yeast that is responsive to physiological stress.

Herberg FW, Bell SM, Taylor SS
Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states.
Protein Eng. 1993; 6: 771-7
Display abstract
The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography. These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate. The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E. coli protein kinases. When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840. This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety. The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E. coli is discussed.

Steinberg RA, Cauthron RD, Symcox MM, Shuntoh H
Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.
Mol Cell Biol. 1993; 13: 2332-41
Display abstract
We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharose-conjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine C alpha subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14C]benzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreonine-containing wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.

Ochatt CM, Ulloa RM, Torres HN, Tellez-Inon MT
Characterization of the catalytic subunit of Trypanosoma cruzi cyclic AMP-dependent protein kinase.
Mol Biochem Parasitol. 1993; 57: 73-81
Display abstract
The catalytic subunit of cyclic AMP-dependent protein kinase from Trypanosoma cruzi epimastigote forms was purified by ionic-exchange chromatography, affinity chromatography and sucrose gradient centrifugation. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the purified preparations showed a main polypeptide band with a mobility of about 40 kDa. In Western blots this band immunoreacted with a polyclonal antibody specific for the catalytic subunit of bovine heart protein kinase A. Hydrodynamic and molecular parameters of this subunit are as follows: molecular weight, 40,000 +/- 3000; sedimentation constant, 2.8 +/- 0.3 S; Stokes' radius, 2.8 +/- 0.2 nm; frictional ratio, 1.28 +/- 0.05. Purified preparations of T. cruzi catalytic and regulatory subunits reconstitute a holoenzyme with a sedimentation constant. 8.6 +/- 1.17 S. This data together with those previously reported by Ulloa et al. [8] indicate that the T. cruzi cyclic AMP-dependent protein kinase holoenzyme is a tetramer with the structure R2C2 of about 200 kDa. The apparent Km of the catalytic subunit for ATP and histone IIA or kemptide as phosphate donor and acceptor, respectively, were 40 microM, 48.6 microM and 26 microM, respectively.

Doskeland SO, Maronde E, Gjertsen BT
The genetic subtypes of cAMP-dependent protein kinase--functionally different or redundant?
Biochim Biophys Acta. 1993; 1178: 249-58

Rosenblatt J, De Bondt H, Jancarik J, Morgan DO, Kim SH
Purification and crystallization of human cyclin-dependent kinase 2.
J Mol Biol. 1993; 230: 1317-9
Display abstract
The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent protein kinases. We report the purification and crystallization of the catalytic subunit of human cyclin-dependent kinase 2 (CDK2), which has been implicated in the control of the G1/S transition. CDK2 was purified in large quantities from insect cells infected with a recombinant baculovirus, and crystals of the protein were prepared and subjected to preliminary X-ray diffraction analysis.

Bossemeyer D
Loss of kinase activity.
Nature. 1993; 363: 590-590

Nishida E, Gotoh Y
The MAP kinase cascade is essential for diverse signal transduction pathways.
Trends Biochem Sci. 1993; 18: 128-31
Display abstract
Mitogen-activated protein (MAP) kinases are activated by combined tyrosine and threonine phosphorylation catalysed by MAP kinase kinase, a novel class of protein kinases with dual specificity for both tyrosine and serine/threonine. MAP kinase kinase is turned on by serine/threonine phosphorylation catalysed by an immediate upstream kinase. The MAP kinase cascade appears to be conserved during evolution and thus might play an essential role in diverse intracellular signaling processes from yeasts to vertebrates.

Haystead CM, Gregory P, Sturgill TW, Haystead TA
Gamma-phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase.
Eur J Biochem. 1993; 214: 459-67
Display abstract
Recently, Sowadski and colleagues [Knighton, D.R., Zheng, J., Eyck, L.F.T., Ashford, V.A., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1991) Science 407, 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D.R., Eyck, L.F.T., Karlsson, R., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the alpha, beta and gamma phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [gamma-32P]adenosine-5'-(gamma-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its gamma phosphate. In the presence of 20 microM ATP, adenosine-5'-(gamma-4-aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 microM against cyclic A kinase, mitogen-activated protein kinase (p42mapk), mitogen-activated protein kinase kinase and p60c-src, respectively. To illustrate the effectiveness of adenosine-5'-(gamma-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity.

Otsu M, Terada Y, Okayama H
Isolation of two members of the rat MAP kinase gene family.
FEBS Lett. 1993; 331: 307-307

Tomoda T, Murata T, Arai K, Muramatsu M
Mutations on 170Glu, a substrate recognition residue in mouse cAMP-dependent protein kinase, generate enzymes with altered substrate affinity and biological functions.
Biochim Biophys Acta. 1993; 1175: 333-42
Display abstract
Site-directed mutations in the catalytic subunit of mouse cAMP-dependent protein kinase (PKAcat) were generated to assess the residue(s) important for the recognition of the substrate peptide and its biological functions. Since the region, 165R-166D-167L-168K-169P-170E-171 N of PKAcat has been shown to be located near the substrate analogue inhibitor peptide binding site (Knighton et al. (1991) Science 253, 414-420), we initially constructed three PKAcat mutants, D166A, K168A, and E170A, in which 166D, 168K, and 170E, respectively, were altered to alanine. When expressed in COS7 cells, D166A and K168A were insoluble, whereas E170A was soluble but had lower in-vitro kinase activity than the wild-type PKAcat. E170A and other 170E mutants, E170Q, E170V, E170R and E170D were equally soluble and displayed various catalytic activities with increased Km and decreased Vmax with regard to Kemptide substrate. Most prominently, E170R did not phosphorylate Kemptide, suggesting that 170E is important for the interaction with Kemptide. The in-vivo activities of the PKAcat mutants were examined in two independent biological assays. First, in Jurkat cells, overexpression of all the 170E mutants except E170R activated the c-fos promoter at various levels lower than the wild-type PKAcat, suggesting that these mutants retain at least partial biological activity. Second, progesterone-induced germinal vesicle break-down in Xenopus oocytes, inhibited by expression of wild-type PKAcat, was inhibited to a similar extent by all the 170E mutants except E170R. All these results support the idea that 170E is a peptide-recognition residue.

Keyse SM, Ginsburg M
Amino acid sequence similarity between CL100, a dual-specificity MAP kinase phosphatase and cdc25.
Trends Biochem Sci. 1993; 18: 377-8

Zhang F, Robbins DJ, Cobb MH, Goldsmith EJ
Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag.
J Mol Biol. 1993; 233: 550-2
Display abstract
The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1). The cell constants are a = 49.32 A, b = 71.42 A, c = 61.25 A, and beta = 109.75 degrees, and the crystals diffract to better than 1.8 A resolution.

Yonemoto W, Garrod SM, Bell SM, Taylor SS
Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1993; 268: 18626-32
Display abstract
The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E. coli is due to autophosphorylation. Whether this process is intramolecular or intermolecular cannot be distinguished. Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.

Orellana SA, Amieux PS, Zhao X, McKnight GS
Mutations in the catalytic subunit of the cAMP-dependent protein kinase interfere with holoenzyme formation without disrupting inhibition by protein kinase inhibitor.
J Biol Chem. 1993; 268: 6843-6
Display abstract
Three amino acids were identified in the catalytic (C) subunit of the cyclic AMP-dependent protein kinase that are involved in interaction with regulatory (R) subunit, but not with the specific protein kinase inhibitor, PKI. In a functional assay for gene induction, a C expression vector with serine or arginine substituted for Leu-198 and the double mutant C, His-87-->Gln/Trp-196-->Arg (Orellana, S. A., and McKnight, G. S. (1992) Proc. Natl. Acad. Sci, U.S.A. 89, 4726-4730), retained activity in the presence of an excess of RI or RII. In contrast, cotransfection of a full-length PKI expression vector completely inhibited the activity of both mutant and wild type C subunits. These data suggest that although the substrate/pseudosubstrate sites of R and PKI interact with C at the catalytic site, there is an additional domain on the C subunit that is involved in holoenzyme formation with R subunit and is distinct from sites specifying high affinity PKI binding.

Kwon YG, Srinivasan J, Mendelow M, Pluskey S, Lawrence DS
Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates.
J Biol Chem. 1993; 268: 16725-9
Display abstract
The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers. The threoninol residue contains both a primary alcohol and a secondary alcohol, either of which may serve as a site of phosphorylation. The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a Km of 498 +/- 39 microM and a Vmax of 7.8 +/- 0.2 mumol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a Km of 16.3 +/- 0.8 microM and a Vmax of 16.0 +/- 0.4 mumol/min-mg. Mass spectral analysis of the phosphopeptide reaction products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation between the C-2 stereoisomers is discussed.

Keryer G, Luo Z, Cavadore JC, Erlichman J, Bornens M
Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2.
Proc Natl Acad Sci U S A. 1993; 90: 5418-22
Display abstract
Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins. By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases. The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta. This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date. In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2. The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed.

Adams JA, Taylor SS
Phosphorylation of peptide substrates for the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1993; 268: 7747-52
Display abstract
The steady-state kinetic parameters for the phosphorylation of four peptides by the catalytic subunit of cAMP-dependent protein kinase were measured as a function of pH. For peptides containing the minimum consensus sequence, R-R-X-S-hyd (where hyd is a hydrophobic residue), the kcat/Kpeptide profile is bell-shaped with pK values of 6.4 and 9.4. Inhibition studies with the peptide LRRNAI indicate that the lower pK corresponds to an intrinsic pK on the enzyme, whereas the higher pK is perturbed upward by 1 pH unit. Viscosity studies verify that substrate stickiness accounts for the kinetic perturbation of the higher pK in kcat/Kpeptide. Substitution of the P-3 arginine with alanine (where serine is the P-site) yields a kcat/Kpeptide versus pH profile that is also bell-shaped, although both pK values are intrinsic acid dissociation constants of the enzyme. Replacement of the P-2 arginine with alanine removes the lower pK in the pH-rate profile without altering the higher pK. These results indicate that recognition of the P-2 arginine requires the ionization of an enzyme residue. This result implies that if the catalytic subunit mechanism involves general base catalysis, the ionization of this bse is not manifested in the pH-rate profiles.

Zheng J, Knighton DR, Xuong NH, Taylor SS, Sowadski JM, Ten Eyck LF
Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.
Protein Sci. 1993; 2: 1559-73
Display abstract
Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

Betzel C et al.
Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution.
J Biol Chem. 1993; 268: 15854-8
Display abstract
The crystal structure of a transition state/product complex formed by the interaction between proteinase K and the substrate analogue N-Ac-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2 has been determined at a resolution of 2.2 A and refined to an R-factor of 0.165 for 12,725 reflections. The inhibitor forms a stable complex through a series of hydrogen bonds with protein atoms and water molecules. The inhibitor is hydrolyzed between Phe 4I and D-Ala5I (I indicates inhibitor). The two fragments are separated by a distance of 3.07 A between the carbonyl carbon and the main chain nitrogen. Both fragments remain bound to the protein. The N-terminal fragment occupies subsites S5 to S1, whereas the C-terminal part is bound in S1' and S2', the first time that electron density for a substrate analogue has been observed in the P1' and P2' sites of a subtilisin-like enzyme. The flexible segments of the substrate recognition sites Gly100-Tyr104 and Ser132-Gly136 move appreciably to accommodate the inhibitor. Biochemical results indicate an inhibition by this specifically designed peptide of 95%.

Kwon YG et al.
The active site substrate specificity of the cAMP-dependent protein kinase.
J Biol Chem. 1993; 268: 10713-6
Display abstract
cAMP-dependent protein kinase substrates have been synthesized employing an unusually efficient method that allows the alcohol-bearing residue to be incorporated into the peptide after solid phase peptide synthesis. These peptide substrates have been utilized to map the active site substrate specificity of the protein kinase. Only alpha- or beta-substituted alcohol-bearing residues containing the proper absolute configuration are phosphorylated by the enzyme. However, the cAMP-dependent protein kinase will phosphorylate achiral residues. The implications of the observed protein kinase substrate specificity with respect to inhibitor design are discussed.

Olah GA, Mitchell RD, Sosnick TR, Walsh DA, Trewhella J
Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide.
Biochemistry. 1993; 32: 3649-57
Display abstract
Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

Buechler YJ, Herberg FW, Taylor SS
Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95.
J Biol Chem. 1993; 268: 16495-503
Display abstract
The type I alpha regulatory subunits of cAMP-dependent protein kinase contain an autoinhibitor site, Arg94-Arg-Gly-Ala-Ile, which serves as a pseudosubstrate. To evaluate their contribution to subunit association, Arg94 and Arg95, key determinants for peptide recognition, were replaced singly and in tandem with Ala, Glu, and His. Unlike substrate peptides in which replacement of either arginine leads to an increase in Km of approximately 3 orders of magnitude, replacement of either arginine causes only a maximal 20-fold decrease in subunit association. Replacement of both arginine residues with alanine, however, generates a regulatory subunit that can no longer recombine with the catalytic subunit under physiological conditions when the regulatory subunit is saturated with cAMP. To evaluate more fully the specific consequences of replacing these 2 arginine residues, a rapid gel filtration chromatographic method was developed so that subunit affinity could be measured independently of assaying for catalytic activity. The R94,95A mutant shows a Kd(app) = 677 nM, representing an increase of greater than 3 orders of magnitude compared with the native subunits in the presence of MgATP. In the absence of MgATP, the Kd(app) for native regulatory subunit was 125 nM, whereas the Kd(app) for the R94,R95A mutant regulatory subunit was determined to 2.87 microM. When this mutant holoenzyme is assayed at microM concentrations, no activity is observed, whereas below microM, activity is observed because of cAMP-independent subunit dissociation.

Yonemoto W, McGlone ML, Taylor SS
N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability.
J Biol Chem. 1993; 268: 2348-52
Display abstract
Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.

Robbins DJ et al.
Regulation and properties of extracellular signal-regulated protein kinases 1, 2, and 3.
J Am Soc Nephrol. 1993; 4: 1104-10
Display abstract
The extracellular signal-regulated kinases ERK1 and ERK2 are 43- and 41-kd enzymes activated by many extracellular cues. They lie within a protein kinase cascade that is used to achieve many cellular responses. In addition to the wide variety of regulatory contexts in which they are activated, they phosphorylate important regulatory proteins, including receptors, transcription factors, cytoskeletal proteins, and other protein kinases. Thus, the stimulation of this kinase cascade is thought to have a pleiotropic action. ERK1 and ERK2 are controlled by phosphorylation on threonine and tyrosine. To understand the regulatory mechanisms, wild-type and mutant ERKs were expressed in bacteria and phosphorylated with MEK, the enzyme that is upstream of ERKs. Wild-type proteins could be activated 500- to 1,000-fold in vitro by MEK. ERK3, an enzyme of 62 kd and only 50% identical to ERK1 and ERK2 in the catalytic core, was also phosphorylated by MEK in vitro. This suggests that all three of these enzymes are targets of common signaling pathways.

Anjard C, Etchebehere L, Pinaud S, Veron M, Reymond CD
An unusual catalytic subunit for the cAMP-dependent protein kinase of Dictyostelium discoideum.
Biochemistry. 1993; 32: 9532-8
Display abstract
The cAMP-dependent protein kinase (cAPK) plays an essential role during differentiation and fruit morphogenesis in Dictyostelium discoideum. The presence of an open reading frame on the gene, pkaC (previously named either Dd PK2 or Dd PK3 by different groups), predicts a 73-kDa polypeptide with 54% similarity to the catalytic subunits of cAPKs from other organisms. Using anti-peptide antibodies, we show that the pkaC gene product, PkaC, is a 73-kDa polypeptide. Despite the fact that PkaC is about twice the size of its mammalian counterparts, it possesses all of the properties required of a catalytic subunit. It is physically associated with the regulatory subunit, and this association results in an inhibition of the catalytic activity which is reverted by cAMP. PkaC copurifies with cAPK activity, and an increased cAPK activity is observed in cells overexpressing PkaC. We conclude that PkaC is a catalytic subunit of the Dictyostelium discoideum cAPK and discuss the unusual features of this protein with the highest molecular weight of known cAPKs.

Su Y, Taylor SS, Dostmann WR, Xuong NH, Varughese KI
Crystallization of a deletion mutant of the R-subunit of cAMP dependent protein kinase.
J Mol Biol. 1993; 230: 1091-3
Display abstract
A single deletion (delta 1 to 91) mutant of the regulatory subunit of the cAMP dependent protein kinase was crystallized. The crystals are hexagonal P6(1)22 (P6(5)22) with a = b = 88.7 A and c = 179.9 A. The crystals diffract to 3 A resolution. There is one molecule per asymmetric unit.

Qamar R, Cook PF
pH dependence of the kinetic mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit in the direction of magnesium adenosine 5'-diphosphate phosphorylation.
Biochemistry. 1993; 32: 6802-6
Display abstract
In order to determine the acid-base chemical mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the pH dependence of the kinetic mechanism in the direction of MgADP phosphorylation has been determined using initial velocity studies in the presence and absence of dead-end inhibitors. The kinetic mechanism in the direction of MgADP phosphorylation is random, with changes in the preference of substrate binding as a function of pH. At pH 7.2 and below, the kinetic mechanism is ordered with phosphorylated peptide binding prior to MgADP. At pH 7.6, the opposite pathway with MgADP binding prior to phosphorylated peptide is preferred. The pH independence of V/Et is consistent with a mechanism in which reactants only bind to the correctly protonated form of the enzyme. The V/KMgADP value decreases, as the pH increases, giving a pK of about 7 which is likely that of a general acid, the same group that serves as a general base in the opposite reaction direction [Yoon, M.-Y., & Cook, P.F. (1987) Biochemistry 26, 4118]. The pKiPSP decreases at low pH giving a pK of about 6.5, probably reflecting the phosphate group of the peptide.

Toner-Webb J, van Patten SM, Walsh DA, Taylor SS
Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1992; 267: 25174-80
Display abstract
The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.

Fantozzi DA, Taylor SS, Howard PW, Maurer RA, Feramisco JR, Meinkoth JL
Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1992; 267: 16824-8
Display abstract
cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.

Stehle T, Schulz GE
Refined structure of the complex between guanylate kinase and its substrate GMP at 2.0 A resolution.
J Mol Biol. 1992; 224: 1127-41
Display abstract
The crystal structure of guanylate kinase from Saccharomyces cerevisiae complexed with its substrate GMP has been refined at a resolution of 2.0 A. The final crystallographic R-factor is 17.3% in the resolution range 7.0 A to 2.0 A for all reflections of the 100% complete data set. The final model has standard geometry with root-mean-square deviations of 0.016 A in bond lengths and 3.0 in bond angles. It consists of all 186 amino acid residues, the N-terminal acetyl group, the substrate GMP, one sulfate ion and 174 water molecules. Guanylate kinase is structurally related to adenylate kinases and G-proteins with respect to its central beta-sheet with connecting helices and the giant anion hole that binds nucleoside triphosphates. These nucleotides are ATP and GTP for the kinases and GTP for the G-proteins. The chain segment binding the substrate GMP of guanylate kinase differs grossly from the respective part of the adenylate kinases; it has no counterpart in the G-proteins. The binding mode of GMP is described in detail. Probably, the observed structure represents one of several structurally quite different intermediate states of the catalytic cycle.

Adams JA, Taylor SS
Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments.
Biochemistry. 1992; 31: 8516-22
Display abstract
Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase. The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima. The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively. Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP. Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high. Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated. The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms. The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.

Cheley S, Bayley H
Kinetics and regulation of two catalytic subunits of cAMP-dependent protein kinase from Aplysia californica.
Biochemistry. 1991; 30: 10246-55
Display abstract
CAPL-A1 and CAPL-A2, two catalytic subunits of Aplysia cAMP-dependent protein kinase, are encoded by mRNAs generated by alternative splicing of transcripts of a gene that contains two mutually exclusive exon cassettes. The subunits are identical except for amino acids 142-183 of the 352 residues, which differ at 10 of 42 positions. CAPL-A1 and CAPL-A2 have now been expressed in insect cells and purified to homogeneity. The subunits differ in their catalytic properties, which have been determined with a series of synthetic peptide substrates. For example, kcat and Km values for the peptide LRRASLG (kemptide) are 42 s-1 and 36 microM and 28 s-1 and 17 microM for CAPL-A1 and CAPL-A2, respectively. CAPL-A1 and CAPL-A2 have different substrate specificities. For example, (kcat/Km)peptide-T/(kcat/Km)kemptide is 9.1 x 10(-3) for CAPL-A1 and 15 x 10(-3) for CAPL-A2, where peptide-T is the kemptide homologue LRRATLG. The subunits also differ in regulation as determined by their interactions with a purified type I regulatory subunit, which has an IC50 for CAPL-A1 that is 3.5 times higher than the IC50 for CAPL-A2. These modest differences reinforce accumulating evidence that the physiological state of a cell depends upon a spectrum of protein kinases with overlapping substrate specificities and regulatory properties.

Wu J, Rossomando AJ, Her JH, Del Vecchio R, Weber MJ, Sturgill TW
Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine.
Proc Natl Acad Sci U S A. 1991; 88: 9508-12
Display abstract
Mitogen-activated protein kinase (MAP kinase) is a serine/threonine protein kinase that becomes enzymatically activated and phosphorylated on tyrosine and threonine following treatment of quiescent cells with a variety of stimulatory agonists. Phosphorylation on both tyrosine and threonine is necessary to maintain full activity, and these two regulatory phosphorylations occur close to each other, separated by a single glutamate. To study the mechanisms by which MAP kinase becomes phosphorylated and activated, we have cloned a full-length cDNA encoding MAP kinase and have expressed the enzyme in Escherichia coli as a soluble nonfusion protein. We find that the enzyme displays a basal, intramolecular autophosphorylation on tyrosine-185 that is accompanied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein displays a small fraction of the activity seen with the fully activated, doubly phosphorylated enzyme isolated from mammalian cells but is activated 10- to 20-fold relative to the unphosphorylated enzyme. These findings raise the possibility that regulation of MAP kinase activity in response to agonist stimulation could occur in part through the enhancement of autophosphorylation on tyrosine.

Pattanaik A, Gowda DC, Urry DW
Phosphorylation and dephosphorylation modulation of an inverse temperature transition.
Biochem Biophys Res Commun. 1991; 178: 539-45
Display abstract
Poly[15(IPGVG),(RGYSLG)], where RGYSLG is a protein kinase site, was synthesized. On raising the temperature of a 5 mg/ml solution, this polypeptide undergoes an inverse temperature transition at 18 degrees C in which it folds into a contracted state by optimizing intramolecular hydrophobic interactions. Averaging the data of five experiments, phosphorylation by means of a 3':5' cyclic AMP dependent protein kinase to the extent of one phosphate in 360 residues raises the temperature of the folding transition to 32 degrees C. The shift is completely reversed on dephosphorylation by alkaline phosphatase. Phosphorylation is hereby shown to be the most potent chemical perturbation known for shifting the temperature of an inverse temperature transition, which has been shown to be an efficient mechanism for achieving chemomechanical transduction (mechanochemical coupling).

Knighton DR, Xuong NH, Taylor SS, Sowadski JM
Crystallization studies of cAMP-dependent protein kinase. Cocrystals of the catalytic subunit with a 20 amino acid residue peptide inhibitor and MgATP diffract to 3.0 A resolution.
J Mol Biol. 1991; 220: 217-20
Display abstract
Crystallographic studies of the catalytic subunit of cAMP-dependent protein kinase demonstrate that the presence of a 20 amino acid residue peptide inhibitor and MgATP during crystallization yields crystals with a different space group and, more significantly, makes an important difference in the quality of the resulting crystals. Under identical experimental conditions, the kinase crystallizes in a cubic space group P4(1)32 (a = b = c = 169.24 A), when no substrates or inhibitors are present, and in the hexagonal space group P6(1)22 (or P6(5)22) (a = b = 80.16 A, c = 288.07 A, alpha = beta = 90 degrees, gamma = 120 degrees) when a 20-amino acid residue peptide inhibitor and MgATP are present. Moreover, the hexagonal crystal diffracts to a resolution of 3.0 A, while the cubic crystals diffract to a resolution of 4.0 A.

Seger R et al.
Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation.
Proc Natl Acad Sci U S A. 1991; 88: 6142-6
Display abstract
Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.

Fernandez A, Mery J, Vandromme M, Basset M, Cavadore JC, Lamb NJ
Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.
Exp Cell Res. 1991; 195: 468-77
Display abstract
In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

Tamanini A, Berton G, Cabrini G
Adenosine 3':5'-monophosphate-dependent protein kinase from human placenta: characterization of the catalytic subunit.
Enzyme. 1991; 45: 97-108
Display abstract
The catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37) was purified for the first time from human placenta by DEAE-cellulose and HTP chromatography. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a single band of average molecular weight of 42 kDa (SEM = 0.52). Kinetic experiments showed a Km for ATP of 12.6 +/- 1.2 mumol/l, for histone II-AS of 1.3 +/- 0.05 mg.ml-1, for kemptide of 11.4 +/- 4.4 mumol/l. The synthetic inhibitor IP20-amide showed a competitive mechanism of inhibition with a Ki of 5.0 nmol/l. The protein kinase inhibitors H7 and H9 showed an apparent Ki of 8.3 and 4.9 mumol/l respectively. Preparative isoelectric focusing revealed the presence of 5 different isoforms with an average pI of 6.17, 6.70, 7.15, 7.67, 8.9.

Meyerson M, Faha B, Su LK, Harlow E, Tsai LH
The cyclin-dependent kinase family.
Cold Spring Harb Symp Quant Biol. 1991; 56: 177-86

Payne DM et al.
Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).
EMBO J. 1991; 10: 885-92
Display abstract
Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

Zheng JH, Knighton DR, Parello J, Taylor SS, Sowadski JM
Crystallization of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase.
Methods Enzymol. 1991; 200: 508-21

Flaherty KM, DeLuca-Flaherty C, McKay DB
Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.
Nature. 1990; 346: 623-8
Display abstract
The three-dimensional structure of the amino-terminal 44K ATPase fragment of the 70K bovine heat-shock cognate protein has been solved to a resolution of 2.2 A. The ATPase fragment has two structural lobes with a deep cleft between them; ATP binds at the base of the cleft. Surprisingly, the nucleotide-binding 'core' of the ATPase fragment has a tertiary structure similar to that of hexokinase, although the remainder of the structures of the two proteins are completely dissimilar, suggesting that both the phosphotransferase mechanism and the substrate-induced conformational change intrinsic to the hexokinases may be used by the 70K heat shock-related proteins.

Arora KK, Shenbagamurthi P, Fanciulli M, Pedersen PL
Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP.
J Biol Chem. 1990; 265: 5324-8
Display abstract
A 50-amino acid peptide predicted by chemical modification studies of yeast hexokinase to contain an ATP-binding site has been synthesized and purified. The peptide, which includes residues from glutamate 78 at the NH2-terminal end to leucine 127 at the COOH-terminal, resides within the smaller of the two lobes found in the three-dimensional structure of yeast hexokinase. It is this region which has been reported recently to exhibit significant sequence homology with hexokinase types I and IV of higher eukaryotic cells and sequence homology with the active site of protein kinases. Similar to native yeast hexokinase, the 50-amino acid peptide interacts strongly with the fluorescent analog TNP-ATP [2',(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate]. A 5-fold enhancement is observed when 8 microM peptide interacts with 20 microM TNP-ATP. The stoichiometry of binding is very close to 1 mol of TNP-ATP/mol peptide. Also, similar to native yeast hexokinase, the fluorescent enhancement observed upon TNP-ATP binding to the synthetic peptide is greater than that observed upon TNP-ADP binding. Finally, TNP-AMP exhibits a much lower fluorescent enhancement in the presence of hexokinase or the synthetic peptide. The additional findings that ATP can readily prevent TNP-ATP binding and that TNP-ATP can substitute for ATP as a weak substrate for hexokinase in the phosphorylation of glucose indicate that the synthetic peptide described here comprises part of the catalytic site.

Salerno A, Mendelow M, Prorok M, Lawrence DS
Noncovalent active site interactions enhance the affinity and control the binding order of reversible inhibitors of the cAMP-dependent protein kinase.
J Biol Chem. 1990; 265: 18079-82
Display abstract
The peptides, Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH2, Leu-Arg-Arg-Gln-Ala-Leu-Gly-NH2, and Leu-Arg-Arg-Asn-Ala-Leu-Gly-NH2, serve as active site-directed inhibitors of the cAMP-dependent protein kinase from bovine cardiac muscle. The Asn-containing peptide is a 10-fold more potent inhibitor than its Ala- and Gln-containing counterparts. All three peptides are linear competitive inhibitors versus a peptide-based substrate and uncompetitive inhibitors versus MgATP. The enhanced inhibitory potency of the Asn-peptide, in conjunction with the observed loss of ATP-ase activity of the enzyme in the presence of the inhibitor, suggests that asparagine may serve as a through-space isostere of serine. The uncompetitive inhibition pattern displayed by amide-capped peptides versus MgATP indicates that these species bind in an ordered fashion to the cAMP-dependent protein kinase, with MgATP binding first.

Mobashery S, Doughty M, Kaiser ET
Inactivation of the catalytic subunit of bovine cAMP-dependent protein kinase by a peptide-based affinity inactivator.
Biopolymers. 1990; 29: 131-8
Display abstract
A peptide affinity inactivator, Ac-Leu-Arg-Arg-Ala-(BrAc)Orn-Leu-Gly, was used as a tool to probe for active site residues in the catalytic subunit of bovine cAMP-dependent protein kinase. The peptide inactivated the catalytic subunit in an active site-directed and monophasic manner with a first-order rate constant of 0.03 min-1 and a dissociation constant of 675 microM. Studies with radioactive peptide indicated that approximately one equivalent of peptide was incorporated into each protein molecule. Protein sequencing identified the modified residue as Cys-199. A possible location for Cys-199 within the active site is suggested.

Ringheim GE, Taylor SS
Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP.
J Biol Chem. 1990; 265: 4800-8
Display abstract
A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.

Hall FL, Braun RK, Mitchell JP, Vulliet PR
Phosphorylation of cytoskeletal proteins by proline directed protein kinase.
Proc West Pharmacol Soc. 1990; 33: 213-7

Buechler JA, Taylor SS
Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides.
Biochemistry. 1990; 29: 1937-43
Display abstract
The catalytic subunit of cAMP-dependent protein kinase typically phosphorylates protein substrates containing basic amino acids preceding the phosphorylation site. To identify amino acids in the catalytic subunit that might interact with these basic residues in the protein substrate, the enzyme was treated with a water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), in the presence of [14C]glycine ethyl ester. Modification of the catalytic subunit in the absence of substrates led to the irreversible, first-order inhibition of activity. Neither MgATP nor a 6-residue inhibitor peptide alone was sufficient to protect the catalytic subunit against inactivation by the carbodiimide. However, the inhibitor peptide and MgATP together completely blocked the inhibitory effects of EDC. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and [14C]glycine ethyl ester. After purification and sequencing, these carboxyl groups were identified as Glu 107, Glu 170, Asp 241, Asp 328, Asp 329, Glu 331, Glu 332, and Glu 333. Three of these amino acids, Glu 331, Glu 107, and Asp 241, were labeled regardless of the presence of substrates, while Glu 333 and Asp 329 were modified to a slight extent only in the free catalytic subunit. Glu 170, Asp 328, and Glu 332 were all very reactive in the apoenzyme but fully protected from modification by EDC in the presence of MgATP and an inhibitor peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Lebioda L, Hargrave PA, Palczewski K
X-ray crystal structure of sangivamycin, a potent inhibitor of protein kinases.
FEBS Lett. 1990; 266: 102-4
Display abstract
The X-ray crystal structure of sangivamycin, a potent nucleoside inhibitor of protein kinases, has been determined. Sangivamycin crystallizes from water with its purine ring in a conformation anti to its ribose sugar. Such an anti conformation has been detected in solution for sangivamycin and other potent protein kinase inhibitors and appears to correlate with inhibitor potency [(1990) Biochemistry (in press)]. An intramolecular hydrogen bond between purine ring substituents is detected in the X-ray structure and may be an important structural feature of sangivamycin related to its degree of inhibition of rhodopsin kinase and of protein kinases C and A.

Jaskolski M, Miller M, Rao JK, Leis J, Wlodawer A
Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.
Biochemistry. 1990; 29: 5889-98
Display abstract
The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

Taylor SS
cAMP-dependent protein kinase. Model for an enzyme family.
J Biol Chem. 1989; 264: 8443-6

Mildvan AS
Nuclear relaxation and Overhauser effect studies of enzyme-substrate interactions.
Arch Biol Med Exp (Santiago). 1989; 22: 147-51

Buechler JA, Taylor SS
Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase.
Biochemistry. 1989; 28: 2065-70
Display abstract
In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.

Prorok M, Lawrence DS
Intrasubstrate steric interactions in the active site control the specificity of the cAMP-dependent protein kinase.
Biochem Biophys Res Commun. 1989; 158: 136-40
Display abstract
The cAMP-dependent protein kinase catalytic subunit phosphorylates serine residues more efficiently than threonine residues in synthetic peptides. In marked contrast, both amino acids are phosphorylated at similar rates when contained within the appropriate intact protein substrate. The structural basis for the discriminatory behavior observed in small peptides has been investigated and found to be a result of intrapeptide steric interactions in the vicinity of the threonine alcohol moiety. Leu-Arg-Arg-Gly-Thr-Leu-Gly, which is nearly free of these interactions, is phosphorylated at a rate that is almost comparable to its serine-containing counterpart.

First EA, Johnson DA, Taylor SS
Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase.
Biochemistry. 1989; 28: 3606-13
Display abstract
The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Cohen FE, Gregoret L, Presnell SR, Kuntz ID
Protein structure predictions: new theoretical approaches.
Prog Clin Biol Res. 1989; 289: 75-85

Buechler JA, Vedvick TA, Taylor SS
Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with acetic anhydride: substrate-induced conformational changes.
Biochemistry. 1989; 28: 3018-24
Display abstract
In order to identify regions that are sensitive to substrate-induced perturbations, the catalytic subunit of cAMP-dependent protein kinase was differentially labeled with [3H]acetic anhydride. Treatment of the catalytic subunit with acetic anhydride in the absence of substrates led to the irreversible inhibition of activity, and MgATP protected against inactivation. After development of a purification protocol for the lysine-containing peptides, the reactivity of each lysine in the native enzyme was calculated. The reactivity profile of lysines in the apoenzyme revealed three distinct regions. In general, the lysines within the amino-terminal segment (residues 1-83) and the carboxy-terminal segment (192-345) were relatively reactive. In contrast, the five lysines in the middle of the protein (Lys-92, -105, -111, -168, and -189) were very unreactive, indicating that these groups are sequestered from the aqueous solvent. The reactivity of each lysine was then determined in the presence of MgATP and in the presence of MgATP and a 20-residue inhibitor peptide. Most of the substrate-induced changes in lysine reactivity were localized in the amino-terminal segment, while the reactivities of lysines in the carboxy-terminal region were not altered significantly by MgATP or inhibitor peptide. MgATP affords substantial protection to three residues in particular. Lys-72, predicted previously to be essential for nucleotide binding was relatively reactive in the apoenzyme, whereas labeling was nearly abolished in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Glass DB, Lundquist LJ, Katz BM, Walsh DA
Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase.
J Biol Chem. 1989; 264: 14579-84
Display abstract
The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.

Glass DB, Cheng HC, Mende-Mueller L, Reed J, Walsh DA
Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein.
J Biol Chem. 1989; 264: 8802-10
Display abstract
PKI-(5-24)-amide is a 20-residue peptide with the sequence, Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-A la-Ile-His- Asp24-NH2, that corresponds to the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992). Amino acid residues in PKI-(5-24)-amide responsible for the potent inhibition (Ki = 2.3 nM) of the catalytic subunit of protein kinase were further investigated using deletion and substitution analogs of the synthetic peptide. Residues 5, 23, and 24 were not required for activity since the 17-residue PKI-(6-22)-amide retained full potency. Sequential removal of the first seven amino acids from the NH2 terminus of PKI-(5-24)-amide caused a progressive 50-fold loss of inhibitory potency. In contrast, substitution of either Thr6, Asp9, or Ile11 with alanine, or Ala8 by leucine, in PKI-(5-22)-amide produced less than 3-fold decreases in potency. Of the 2 aromatic residues in PKI-(5-22)-amide, the individual substitution of Phe10 and Tyr7 by alanine caused, respectively, 90- and 5-fold decreases in inhibitory potency, demonstrating important roles for each. This NH2-terminal portion of the peptide is believed to contain a significant portion of alpha-helix. Many recognition or structural determinants are also essential in the COOH-terminal portion of PKI-(5-22)-amide. In addition to the basic subsite provided by the three arginines, several other of the residues are critical for full inhibitory potency. Substitution of Ile22 by glycine in either PKI-(5-22)-amide or PKI-(14-22)-amide lowered the inhibitory potency by 150- and 50-fold, respectively. Separate replacement of Gly17 or Asn20, in either PKI-(5-22)-amide or PKI-(14-22)-amide, caused 7-15-fold decreases in potency. Substitution of both Gly17 and Asn20 together (in PKI-(14-22)-amide) produced a synergistic loss of inhibitory activity. [Leu13,Ile14]PKI-(5-22)-amide, a doubly substituted analog exhibited a 42-fold increase in Ki value. We conclude that Ser13 and/or Gly14, Gly17, Asn20, and Ile22 each contribute important features to the binding of these inhibitory peptides to the protein kinase, either by providing recognition determinants, inducing structure, and/or allowing essential peptide backbone flexibility.(ABSTRACT TRUNCATED AT 400 WORDS)

McKnight GS et al.
Analysis of the cAMP-dependent protein kinase system using molecular genetic approaches.
Recent Prog Horm Res. 1988; 44: 307-35

Levin LR et al.
A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation.
Science. 1988; 240: 68-70
Display abstract
A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.

Buechler JA, Taylor SS
Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase.
Biochemistry. 1988; 27: 7356-61
Display abstract
The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Bubis J, Vedvick TS, Taylor SS
Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I.
J Biol Chem. 1987; 262: 14961-6
Display abstract
The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.

Toner-Webb J, Taylor SS
Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide.
Biochemistry. 1987; 26: 7371-8
Display abstract
The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase (EC 2.7.1.3) in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent Ki of 60 microM. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD included (1) modification with [14C]DCCD, (2) modification by DCCD in the presence of [3H]aniline, and (3) modification with DCCD and [14C]glycine ethyl ester. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Hagiwara M, Inagaki M, Hidaka H
Specific binding of a novel compound, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) to the active site of cAMP-dependent protein kinase.
Mol Pharmacol. 1987; 31: 523-8
Display abstract
The interaction of the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the most potent and selective inhibitor toward cyclic nucleotide-dependent protein kinases in the series of isoquinolinesulfonamide derivatives, was studied. The addition of H-8 protected the catalytic subunit of the enzyme in a dose-dependent manner from irreversible inactivation by the ATP analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The inactivation followed pseudo-first order kinetics and H-8 reduced the steady state constant of inactivation (Ki) without any effect on the first order rate constant (K3). The quantitative binding of H-8 to the enzyme was measured under conditions of thermodynamic equilibrium using a gel filtration method. The catalytic subunit bound approximately 1 mol of drug/mol of protein with apparent half-maximal binding at 1.0 microM drug, whereas the enzyme irreversibly modified by FSBA did not bind the drug, confirming that the enzyme has no site for H-8 in the catalytic subunit other than the active site. The binding studies also showed that H-8 does not require divalent cations such as Mg2+ to bind to the catalytic subunit of the protein kinase. The binding of H-8 to the active site was characterized using FSBA and other affinity labeling reagents which have been postulated to modify residues at or near the active site of the catalytic subunit. H-8 protected the enzyme against inactivation by FSBA and Cibacron Blue F3GA but did not afford any protection against the covalent modification of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), suggesting that the binding site of H-8 does not involve the gamma-subsite of the ATP binding site in the catalytic subunit, since DTNB and NBD-Cl are thought to modify the residues complementary to gamma-phosphate of the ATP molecules.

Bramson HN, Thomas NE, Miller WT, Fry DC, Mildvan AS, Kaiser ET
Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase.
Biochemistry. 1987; 26: 4666-70
Display abstract
Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.

Sahal D, Fujita-Yamaguchi Y
Protein kinase assay by paper-trichloroacetic acid method: high performance using phosphocellulose paper and washing an ensemble of samples on flat sheets.
Anal Biochem. 1987; 167: 23-30
Display abstract
Phosphocellulose paper has been found to be the paper of choice in assaying protein kinase activities using [gamma-32P]ATP by the trichloroacetic acid method of precipitation and washing. A study of binding of ATP of increasing concentrations at constant specific activity with Whatman 3MM or ATP-coated Whatman 3 MM papers (in vogue) versus phosphocellulose paper (proposed here) has shown that the latter has the least affinity for ATP when washing is done with either trichloracetic acid or trichloroacetic acid containing pyrophosphate. In an experiment where the placental cytosolic protein kinase was serially diluted, the phosphocellulose paper was found to give higher signal/noise ratios at all dilutions studied compared to the other two papers. With regard to the technological side of washing the papers, we have found that the traditional method of cutting papers into small squares before loading the samples is perhaps not the best. Instead, we propose the use of a flat sheet matrix for loading the samples because this method ensures uniformity of washing among the samples while shaking is performed on a simple shaker. In addition, the whole paper matrix can provide an almost instantaneous autoradiogram of hundreds of samples facilitating biochemical experimentation with protein kinases.

Reed J, Kinzel V, Cheng HC, Walsh DA
Circular dichroic investigations of secondary structure in synthetic peptide inhibitors of cAMP-dependent protein kinase: a model for inhibitory potential.
Biochemistry. 1987; 26: 7641-7
Display abstract
The structure of the inhibitory domain of the inhibitor protein of the cAMP-dependent protein kinase has been assessed by circular dichroism studies of synthetic inhibitory peptides. Using the inhibitory peptide PKI(5-22)amide (Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile22) [Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992] and shorter peptides of this sequence, it has been estimated that this parent peptide is composed of approximately 30% alpha-helix with the remainder being random coil with one beta-turn. The pseudosubstrate arginine cluster (Arg15-Arg19) is within the suggested region of random coil and beta-turn, representing one critical region of binding recognition by the protein kinase. The alpha-helix region proposed between Thr6 and Ile11 likewise contributes to the full biological potency and specificity of the inhibitor peptide and inhibitor protein. The removal of the two N-terminal threonines, for example, causes both a marked conformational change in the peptide and a diminishment by an order of magnitude of inhibitory activity. It is proposed that this alpha-helix region could serve one of several possibilities, including that it may provide a suitable constraint on the Tyr7 such that the hydroxyl is oriented in a position proximal to the pseudosubstrate domain, and/or may allow the optimal location of other protein kinase recognition signals. These data provide an initial description of some of the structural features of the inhibitor protein that could contribute to its high biological potency.

Scott JD, Glaccum MB, Fischer EH, Krebs EG
Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase.
Proc Natl Acad Sci U S A. 1986; 83: 1613-6
Display abstract
The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser- Ser-Ala . In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The results show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 microM) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or alpha-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.

Van Patten SM, Fletcher WH, Walsh DA
The inhibitor protein of the cAMP-dependent protein kinase-catalytic subunit interaction. Parameters of complex formation.
J Biol Chem. 1986; 261: 5514-23
Display abstract
The formation of a complex between the catalytic subunit of the cAMP-dependent protein kinase and the Inhibitor Protein of this enzyme has been examined by means of nondenaturing gel electrophoresis. Two forms of complex were identified, both containing a 1:1 molar ratio of the component proteins. The formation of the major of the two forms is markedly enhanced by the presence of nucleotide triphosphate and divalent cation. Either Mg2+ or Mn2+ serves to promote complex formation. With Mg2+, only ATP is effective for enhancing complex formation, whereas with Mn2+ complex formation occurs to an equal extent with ATP, GTP, ITP, and adenyl-5'-yl imidodiphosphate. The formation of the two complexes is only minimally dependent upon nucleotide triphosphate. It is suggested that the two types of complex are a result of different species of catalytic subunit. Two principal forms of the complex have been detected occurring maximally in approximately a 2.5:1 ratio. In the accompanying paper (Fletcher, W.H., Van Patten, S.M., Cheng, H-C., and Walsh, D.A. (1986) J. Biol. Chem. 261, 5504-5513), we have described the use of a fluoresceinated derivative of catalytic subunit as a cytochemical probe to localize the Inhibitor Protein and the regulatory subunit of the protein kinase. The integrity of this fluorophore has been further characterized using the method of examining catalytic subunit-Inhibitor Protein interaction delineated here.

Showers MO, Maurer RA
A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1986; 261: 16288-91
Display abstract
While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.

Cheng HC et al.
A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase.
J Biol Chem. 1986; 261: 989-92
Display abstract
As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.

Kondo H, Kinoshita J, Matsuba T, Sunamoto J
Configuration of the active Mg-ATP complex in protein kinase C reaction.
FEBS Lett. 1986; 201: 94-6
Display abstract
To probe the active site structure of protein kinase C stereochemical studies were carried out by using ATP beta S. The enzyme utilizes either one of the diastereomers (SP and RP) of ATP beta S almost equally well as a substrate. This result contrasts with that for cyclic AMP-dependent protein kinase, suggesting that the topography of the nucleotide-binding site is significantly different between the two kinases.

Mildvan AS, Rosevear PR, Fry DC, Bramson HN, Kaiser ET
NMR studies of the mechanism of action and regulation of protein kinase.
Curr Top Cell Regul. 1985; 27: 133-44

Scott JD, Fischer EH, Takio K, Demaille JG, Krebs EG
Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.
Proc Natl Acad Sci U S A. 1985; 82: 5732-6
Display abstract
The amino acid sequence of rabbit skeletal muscle heat-stable inhibitor of the cAMP-dependent protein kinase has been determined by microsequencing techniques. Proof of the structure involved a series of nonoverlapping tryptic fragments for primary identification of 86% of the amino acids. Complementary fragments generated by cleavage with chymotrypsin, Staphylococcus aureus V8 proteinase, and mast cell proteinase II contributed to proof of the structure. The inhibitor is a single polypeptide chain of 75 residues and has a molecular weight of 7829. It lacks tryptophan, proline, and sulfur-containing amino acids. The amino terminus of the inhibitor is blocked by an unidentified group. The amino-terminal region of the molecule contains the kinase inhibitory domain, and synthetic peptides based on the sequence of residues 11-30 are potent competitive inhibitors of the cAMP-dependent protein kinase [Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383]. Residues 14-22 show considerable homology to the "hinge-regions" of the regulatory subunits of the cAMP-dependent protein kinase. The remainder of the molecule shows no similarity to the known amino acid sequence of any protein.

Sowadski JM, Nguyen HX, Anderson D, Taylor SS
Crystallization studies of cAMP-dependent protein kinase. Crystals of catalytic subunit diffract to 3.5 A resolution.
J Mol Biol. 1985; 182: 617-20
Display abstract
The catalytic subunit of cAMP-dependent protein kinase from porcine heart has been crystallized in several different crystal forms. One of these forms diffracts to 3.5 A resolution. It is in monoclinic space group P2(1) with a = 64.24 A, b = 143.58 A, c = 48.40 A, alpha = gamma = 90 degrees and beta = 106.9 degrees.

Puri RN, Bhatnagar D, Roskoski R Jr
Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde.
Biochemistry. 1985; 24: 6499-508
Display abstract
The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.

Kochetkov SN, Abduragimov AR, Gabibov AG, Severin ES
[Mechanism of action of cAMP-dependent protein kinase. V. Free energy spectra]
Mol Biol (Mosk). 1985; 19: 1562-8
Display abstract
The catalytic subunit of cAMP-dependent protein kinase from pig brain was shown to catalyse an isotope exchange reaction ATP in equilibrium with ADP. The kinetic parameters of the exchange were determined. The enzyme can also use GTP as the donor substrate; phosphotransferase and "GTPase" reactions were investigated. Based on the kinetic data obtained in this and in the previous paper the free energy profiles of protein kinase catalysed reactions are discussed.

Titani K et al.
Amino acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3',5'-phosphate dependent protein kinase.
Biochemistry. 1984; 23: 4193-9
Display abstract
The complete amino acid sequence of the regulatory subunit of type I cAMP-dependent protein kinase from bovine skeletal muscle is presented. The S-carboxymethylated protein was cleaved with cyanogen bromide to provide a complete set of nonoverlapping fragments. These fragments were overlapped and aligned by using peptides generated by proteolytic cleavage. The protein contains 379 amino acid residues corresponding to a molecular weight of 42 804. As in the type II regulatory subunit of cAMP-dependent protein kinase, a pattern of internal gene duplication is observed, which is consistent with two cAMP-binding domains. The two types of regulatory subunit from type I and type II kinase display similarities in domain substructure and in amino acid sequence, which provide a molecular basis for new insight into their regulatory roles. Detailed analyses of the homology of the regulatory subunits of type I and type II cAMP-dependent protein kinase and of similar relationships to cGMP-dependent protein kinase and Escherichia coli catabolite gene activator protein are presented in accompanying reports from this laboratory [Takio, K., Smith, S. B., Krebs, E. G., Walsh, K., & Titani, K. (1984) Biochemistry (second paper of three in this issue); Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry (third paper of three in this issue)].

Kochetkov CN, Bagirov EM, Gabirov AG, Severin ES
[Mechanism of action of cAMP-dependent protein kinase. III. Probable role of an enzyme carboxyl group]
Mol Biol (Mosk). 1984; 18: 704-11
Display abstract
By the method of chemical modification the active site of cAMP-dependent pig brain protein kinase was shown to contain a carboxyl group. The kinetic parameters of irreversible inhibition of the enzyme by water-soluble carbodiimide in the presence of ethyl ester of glycine were determined. The ionized carboxyl group-containing amino acid residue found is evidently localized in the immediate proximity to the reaction center. The comparison of the reaction rates of phosphate transfer from the intermediate phosphoform of the enzyme under native and denatured conditions suggests that this residue acts as the general base catalyst of this process.

Mildvan AS, Kaiser ET, Rosevear PR, Bramson HN
NMR studies of the mechanism of cyclic AMP-dependent protein kinase.
Fed Proc. 1984; 43: 2634-9
Display abstract
NMR has been used to study the role of the divalent cation, the conformations, arrangement, and exchange rates of the enzyme-bound metal-ATP and peptide substrates, the mechanism of the phosphoryl transfer, and the structure and role of the regulatory subunit on type II cyclic AMP (cAMP)-dependent protein kinase from bovine heart. The active complex consists of an enzyme-ATP-metal bridge in which the metal is beta, gamma coordinated, with delta chirality at P beta, and a torsional angle at the adenine-ribose bond in the high-anti range (x approximately 80 degrees). The bound heptapeptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly is extended in conformation, forming either a coil or, less likely, a beta turn but not an alpha helix or beta sheet. The distance from the gamma-P of bound ATP analogs to the Ser-OH of the bound peptide (5.3 +/- 0.7 A) would permit a metaphosphate or an elongated phosphorane intermediate or transition state. The regulatory subunit (R2) blocks the peptide- or protein-binding site of the catalytic subunit. The 31P chemical shift of cAMP is not greatly altered on binding to R2, but the resonance is broadened to approximately 32 Hz, which indicates no chemical change but marked immobilization of bound cAMP. A narrower (approximately 7 Hz) 31P resonance at 4.44 ppm is assigned to P-serine-95 of R2 because it disappears with catalytic subunit, Mg2+, and an ADP-generating system.

Reimann EM, Titani K, Ericsson LH, Wade RD, Fischer EH, Walsh KA
Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase.
Biochemistry. 1984; 23: 4185-92
Display abstract
The complete amino acid sequence of the catalytic subunit (gamma subunit) of rabbit skeletal muscle phosphorylase b kinase was determined. The gamma subunit was purified by gel filtration in acidic 8 M urea after reduction and S-carboxymethylation in 7 M guanidine hydrochloride. Cleavage of the gamma subunit at arginyl bonds gave a complete set of nonoverlapping peptides. Overlapping peptides were obtained by cleavage at methionyl, tryptophanyl, or glutamyl bonds and by selected subdigestion of two large peptides obtained by cleavage at methionyl bonds. Sequence analysis established that the protein contains 386 residues corresponding to a molecular weight (Mr) of 44673. Comparison of the gamma subunit with the catalytic subunit of bovine cAMP-dependent protein kinase and with tyrosine-specific kinases of viral origin revealed a significant degree of sequence identity among all of these proteins. These data suggest that calcium-dependent protein kinases may share a common ancestral gene and a common structural basis for catalytic function with a wide variety of other protein kinases which respond to different signals and control quite different processes.

Flockhart DA, Freist W, Hoppe J, Lincoln TM, Corbin JD
ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase.
Eur J Biochem. 1984; 140: 289-95
Display abstract
The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.

Takio K, Smith SB, Krebs EG, Walsh KA, Titani K
Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase.
Biochemistry. 1984; 23: 4200-6
Display abstract
Evidence is presented that establishes the amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle. Complementary sets of overlapping peptides were generated primarily by tryptic digestion and by chemical cleavage at methionyl residues. The analysis was augmented by chemical cleavage at a single tryptophanyl residue and at three of the four aspartyl-proline bonds. Several large fragments generated by limited proteolysis contributed to the proof of structure. The subunit is a single chain of 400 residues corresponding to a molecular weight of 45 004. An amino-terminal segment of about 100 residues is believed to include the region responsible for oligomeric association. The remainder of the molecule consists of two tandem homologous domains, each of which is thought to bind a single molecule of cAMP. Comparison of the three domains with corresponding regions of the type I isozyme, of the Escherichia coli catabolite gene activator protein, and of cGMP-dependent protein kinase indicates extensive regions of homology and as much as 50% identity with the sequence of an internal segment of the type I isozyme.

Bhatnagar D, Hartl FT, Roskoski R Jr, Lessor RA, Leonard NJ
Adenosine cyclic 3',5'-monophosphate dependent protein kinase: nucleotide binding to the chemically modified catalytic subunit.
Biochemistry. 1984; 23: 4350-7
Display abstract
5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates the catalytic subunit of the adenosine cyclic 3',5'-monophosphate dependent protein kinase isolated from bovine cardiac muscle by covalent modification of lysine-71, whereas 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with cysteines-199 and -343 to inactivate the enzyme. All three of these reagents have been postulated to modify residues at or near the active site of the catalytic subunit. ATP (2 mM) in the presence of excess Mg2+ (10 mM) protects the enzyme against inactivation by these reagents. AMP did not afford any protection, but adenosine slightly decreased the rate of inactivation. The specific effects of covalent modification of lysine-71 and cysteines-199 and -343 on nucleotide binding were characterized by fluorescence-polarization titrations with lin-benzoadenine nucleotides as fluorescent ligands. lin-Benzoadenosine is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (38 microM) similar to the Ki for adenosine (35 microM). This value agrees well with the Kd (32 microM) for adenosine determined by fluorescence-polarization titrations. lin-Benzoadenosine 5'-diphosphate (lin-benzo-ADP) has been shown to be a competitive inhibitor with respect to ATP [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], and lin-benzoadenosine 5'-triphosphate (lin-benzo-ATP) is a substrate for the phosphotransferase activity of the protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Risnik VV, Gusev NB
Some properties of the nucleotide-binding site of troponin T kinase-casein kinase type II from skeletal muscle.
Biochim Biophys Acta. 1984; 790: 108-16
Display abstract
Investigation of properties of skeletal muscle troponin T kinase (EC 2.7.1.37) has revealed that the enzyme belongs to the group of casein kinases of the second type. The enzyme consists of two subunits with apparent molecular weights of 44 000 and 26 000 and contains a protein with molecular weight of 39 000, which is probably the proteolytic fragment of the 44 000 subunit. The substrate specificity of troponin T kinase was tested, using 20 analogs of the nucleotide. The enzyme has a low substrate specificity toward the purine base and uses both ATP and GTP as substrates. Modification of the ribose ring does not influence the enzyme interaction with the nucleotide; however, the cleavage of ribose leads to a decrease of the enzyme-nucleotide interaction. Elimination of the gamma-terminal phosphate or its modification by bulky hydrophobic radicals do not affect this interaction. A comparison of the Ki values for different analogs suggests that the interaction of troponin T kinase with the nucleotide occurs via the binding of the purine base and the beta-phosphate group of the analog.

Bramson HN, Kaiser ET, Mildvan AS
Mechanistic studies of cAMP-dependent protein kinase action.
CRC Crit Rev Biochem. 1984; 15: 93-124
Display abstract
The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Reed J, Kinzel V
Ligand binding site interaction in adenosine cyclic 3',5'-monophosphate dependent protein kinase catalytic subunit: circular dichroic evidence for intramolecular transmission of conformational change.
Biochemistry. 1984; 23: 968-73
Display abstract
Blue Dextran has been shown to interact specifically with the nucleotide binding site of the catalytic subunit of cAMP-dependent protein kinase. By observing changes in the induced dichroism associated with the absorbance of the bound chromophore, one can monitor conformational changes in the immediate vicinity of the ATP binding site. With this technique, it has been possible to demonstrate that attachment of ligand at the protein substrate binding site of the enzyme results in a conformational change at the ATP binding site. This alteration takes place in at least two steps, one of which appears to be dependent on the presence of a phosphorylatable hydroxyl group on the substrate and the other being triggered by the "basic subsite" (usually one or more arginine residues) to the N-terminal side of the target serine or threonine. Competition experiments suggest that the change induced results in closure over the substrate protein after the initial electrostatic binding; the movement initiated by the presence of a serine hydroxyl group may also involve interaction with a tyrosine residue at the surface of the ATP binding site.

Whitehouse S, Feramisco JR, Casnellie JE, Krebs EG, Walsh DA
Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase.
J Biol Chem. 1983; 258: 3693-701
Display abstract
The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.

Chessa G, Borin G, Marchiori F, Meggio F, Brunati AM, Pinna LA
Synthetic peptides reproducing the site phosphorylated by cAMP-dependent protein kinase in protein phosphatase inhibitor-1. Effect of structural modifications on the phosphorylation efficiency.
Eur J Biochem. 1983; 135: 609-14
Display abstract
The hexapeptide (Arg)2-Pro-Thr-Pro-Ala (T1) and octapeptide (Arg)2-Pro-Thr-Pro-Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor-1, a physiological target of cAMP-dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP-dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2-Ala-Ser-Val-Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C-terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N-terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T1 on its C-terminal side to give the derivative (Arg)2-Pro-Thr-Pro-Ala-Thr-Val-Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor-1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.

Glass DB
Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors.
Biochem J. 1983; 213: 159-64
Display abstract
The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.

Bagirov EM, Kochetkov SN, Gabibov AG, Sashchenko LP, Severin ES
[Mechanism of action of cAMP-dependent protein kinase. I. Study of pH and temperature dependences of interaction of the catalytic subunit of the enzyme with ATP]
Mol Biol (Mosk). 1983; 17: 129-35

Malencik DA, Anderson SR
Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase.
Anal Biochem. 1983; 132: 34-40
Display abstract
A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.

Shoji S, Ericsson LH, Walsh KA, Fischer EH, Titani K
Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase.
Biochemistry. 1983; 22: 3702-9
Display abstract
The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.

Taylor SS, Kerlavage AR, Zoller MJ
Affinity labeling of cAMP-dependent protein kinases.
Methods Enzymol. 1983; 99: 140-53

Whitehouse S, Walsh DA
Mg X ATP2-dependent interaction of the inhibitor protein of the cAMP-dependent protein kinase with the catalytic subunit.
J Biol Chem. 1983; 258: 3682-92
Display abstract
The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase has been investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was shown to be competitive versus peptide substrate and uncompetitive versus Mg X ATP2-. This, together with the observations of Gronot et al. (Gronot, J., Mildvan, A.S., Bramson, H. N., Thomas, N., and Kaiser, E.T. (1981) Biochemistry 20, 602-610) and those given in the accompanying paper (Whitehouse, S., Feramisco, J.R., Casnellie, J.E., Krebs, E.G., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3693-3701), would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg X ATP complex. The Ki for this interaction at saturating Mg X ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5'-adenylimidodiphosphate (AMP X PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP X PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit, and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 approximately 60 nM for ATP; this value is two orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP but may simply be a reflection of the formation of a catalytic subunit-Mg X ATP X inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.

Conti M, Adamo S, Geremia R, Monesi V
Developmental changes of cyclic adenosine monophosphate-dependent protein kinase activity during spermatogenesis in the mouse.
Biol Reprod. 1983; 28: 860-9

Meggio F, Donella-Deana A, Pinna LA
Heparin, a powerful inhibition of type II casein kinases, stimulates the phosphorylation of some protein substrates by the catalytic subunit of cAMP-dependent protein kinase.
Biochem Int. 1983; 6: 427-32
Display abstract
Heparin, which has been shown to behave as a very effective and specific inhibitor of type II casein kinases, exhibits a stimulatory effect on the phosphorylation rate of pyruvate kinase and phosphorylase kinase, but not of histones, by the catalytic subunit of cAMP-dependent protein kinase. When pyruvate kinase is the substrate the phosphorylation rate is approximately doubled by heparin concentrations around 100 micrograms/ml, but just 2 to 4 micrograms heparin per ml are sufficient to induce a half maximal effect. No stimulation by heparin can be observed replacing the protein substrates with two synthetic peptides reproducing the phosphorylatable sites of pyruvate kinase and of the gamma subunit of phosphorylase kinase. These data support the hypothesis that heparin accelerates phosphorylation by rendering the phosphorylatable sites more readily accessible to the protein kinase.

Takio K, Smith SB, Krebs EG, Walsh KA, Titani K
Primary structure of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle.
Proc Natl Acad Sci U S A. 1982; 79: 2544-8
Display abstract
The complete amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle is presented. Primary fragments for the sequence determination were obtained by limited proteolysis with various proteases or by cleavage with cyanogen bromide. The sequence of the 400 amino acid residues has two homologous regions, strongly suggesting tandem gene duplication. The predicted secondary structure suggests the presence of 42% alpha-helix, 23% beta-strand, and 23 beta-turns. The molecular weight of the subunit, as derived from the sequence, is 45,084 including a phosphate group at residue 95. This is significantly less than earlier estimates based on NaDodSO4 gel electrophoresis and sedimentation experiments. The structure is discussed in terms of putative sites of interaction with cAMP and with the catalytic subunit.

Baydoun H, Hoppe J, Freist W, Wagner KG
Purification and ATP substrate site of a cyclic nucleotide-independent protein kinase (NI) from porcine liver nuclei.
J Biol Chem. 1982; 257: 1032-6

Tang FY, Catapano LS
cAMP-dependent protein kinase II interactions with nuclei derived from rat mammary tumor.
Endocr Res Commun. 1982; 9: 155-68
Display abstract
cAMP-dependent protein kinase from bovine heart exhibited significant interactions with nuclei from rat mammary tumors. These enzyme-nuclear interactions occurred when the intact holoenzyme was preincubated with 10 nM cAMP, 1 mM MgATP at 24 degrees C to produce high-affinity monophasic cAMP-dissociation kinetics. The enzyme-nuclear interactions are correlated with the loss of cAMP and PO4 from the purified enzyme. The data indicate that the high affinity cAMP-dependent protein kinase II exhibits significant nuclear interaction which may be related to cAMP function in rat mammary tumors.

Rymond M, Hofmann F
Characterization of phosphorylated and dephosphorylated regulatory subunit of cAMP-dependent protein kinase II.
Eur J Biochem. 1982; 125: 395-400

Flockhart DA, Corbin JD
Regulatory mechanisms in the control of protein kinases.
CRC Crit Rev Biochem. 1982; 12: 133-86

Ranganathan NS, Linn TC, Srere PA
Phosphorylation of dephospho-ATP citrate lyase by the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1982; 257: 698-702
Display abstract
The native form of ATP citrate lyase (2 mol of phosphate/tetramer) and the dephospho-ATP citrate lyase (phosphate-free) purified to homogeneity from rat liver, are phosphorylated by ATP and by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle. A total of 2 mol of phosphate/tetramer were incorporated into native enzyme, while with the dephospho form, 4 mol of phosphate were incorporated. The phosphopeptides resulting from trypsin treatment which were isolated from phosphorylated forms of both native enzyme and the dephospho enzyme were similar. The ATP citrate lyase, phosphorylated to an extent of 4 mol of phosphate/tetramer, has the same Vmax as the native enzyme (2 mol of phosphate/tetramer). Native ATP citrate lyase, trypsin-treated to remove the phosphopeptide, could not be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase from rabbit muscle, suggesting a common trypsin-sensitive specific phosphorylation site. The phosphorylation rate varied with pH in potassium phosphate, imidazole/HCl, and Tris/HCl buffers. Divalent cations were essential for the activity of the protein kinase. The apparent Km value for ATP was found to be 50 microM.

Baranova LA, Grivennikov IA, Guliaev NN
[Interaction of N1-, N6- and C8-substituted derivatives of adenosine-5'-triphosphate with the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscles]
Biokhimiia. 1982; 47: 1806-13
Display abstract
In order to investigate the structure of the active site of the cAMP-dependent protein kinase catalytic subunit a synthesis of several previously unknown adenosine-5'-triphosphate (ATP) derivatives containing substituents of various nature at N(1), N(C6) and C(8) positions of the purine base was carried out. The interaction of these derivatives with a homogeneous preparation of the catalytic subunit of rabbit skeletal muscle cAMP-dependent protein kinase was investigated. All the nucleotide analogs were found to inhibit the enzyme activity; the inhibition was competitive with respect to ATP. It was assumed that the adenine moiety of the ATP molecule is bound to the active site of protein kinase by the hydrophobic interaction with the aromatic amino acid residues and by formation of the hydrogen bond between the exo-NH2-group of the substrate and a corresponding group of the enzyme. The "correct" binding of ATP to the enzyme active center is defined by the anti-conformation of the nucleotide.

Taylor SS
The in vitro phosphorylation of chromatin by the catalytic subunit of cAMP-dependent protein kinase.
J Biol Chem. 1982; 257: 6056-63
Display abstract
When a mixture of DNA-free core histones (H) from calf thymus is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, phosphate is incorporated primarily into H2B and, to a lesser extent, into H3 and H4. In contrast, when the phosphorylation of the DNA-free histones is compared to the phosphorylation of histones in long chromatin or in nucleosome core, only one of the core histones, H3, is phosphorylated. The site of modification of H3 has been identified as serine 10, which is located in the highly charged basic NH2-terminal region of the molecule. The other sites of phosphorylation in H2B nd H4 are completely masked when the histones are complexed with DNA. It is only when the nucleosome core structure is perturbed that these additional sites become accessible to the kinase. If long chromatin which contains H1 is phosphorylated, histone 1 is also phosphorylated by catalytic subunit at serine 38. However, the addition of excess H1 to H1-depleted long chromatin inhibits the phosphorylation of H3. In addition to the histones, the high mobility group (HMG) protein, HMG 14, was also found to be a good substrate for the kinase. Phosphorylation of HMG 17 in comparison was much less.

Iurkiv VA, Petukhov SP, Bulargina TV, Severin ES
[Isolation and study of the properties of the catalytic subunit of cAMP-dependent protein kinase of the small intestinal mucosa of the rabbit]
Biull Eksp Biol Med. 1981; 91: 429-32
Display abstract
cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.

Kuo WN
Tissue energy of mammalian modulator-dependent protein kinases.
Experientia. 1981; 37: 453-4
Display abstract
The levels of modulator-dependent protein kinases and protamine-dependent protein kinase(s) in various tissues of adult mice were compared. Cerebellum contained the highest levels of both modulator-dependent protein kinases and protamine-dependent protein kinase(s), whereas skeletal muscle contained no detectable enzymes. The lung and the ileum were also rich in modulator-dependent protein kinases, while other tissues were poor sources of these enzymes.

Baydoun H, Hoppe J, Freist W, Wagner KG
The ATP substrate site of a cyclic-nucleotide-independent protein kinase from porcine liver nuclei.
Eur J Biochem. 1981; 115: 385-9
Display abstract
The ATP substrate site of a second messenger-independent protein kinase of the type NII from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP; they range from 4 microM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP-dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into delta delta values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions. With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its beta-phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N-6 amino group is required in the case of the cAMP-dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N-6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.

Bolen DW, Stingelin J, Bramson HN, Kaiser ET
Stereochemical and kinetic studies on the action of the catalytic subunit of bovine cardiac muscle adenosine 3',5'-monophosphate dependent protein kinase using metal ion complexes of ATP beta S.
Biochemistry. 1980; 19: 1176-82

Soumarmon A, Abastado M, Bonfils S, Lewin MJ
C1-transport in gastric micorsomes. An ATP-dependent influx sensitive to membrane potential and to protein kinase inhibitor.
J Biol Chem. 1980; 255: 11682-7
Display abstract
Uptakes of radioactive C1- or 1- by gastric microsomal vesicles were stimulated 2- to 8-fold by AtP. The sensitivity of those uptakes to a C1- in equilibrium OH- ionophore and to osmotic swelling suggested they were due to transport rather than to binding. The ATP effect was labile, but dithiothreitol and methanol improved its stability. The stimulation of anion transport required magnesium; GTP and UTP were less potent than ATP whereas ADP and AMP had no effect. The apparent Km for ATP was estimated to be 2 X 10(-4) M at 22 degrees C. The rate of the ATP-dependent transport showed saturation-type kinetics, with half-maximal uptake at 10 mM for I- and 15 mM for C1-. Nonradioactive C1-, I-, and SCN- competed with 125I- uptake while SO42- did not. K+ valinomycin increased the ATP-dependent C1- uptake. The thermostable inhibitor of cAMP-dependent protein kinases inhibited the effect of ATP. These results suggest the existence of an anion conductance, permeant to C1-, I-, and SCN- and nonpermeant to SO42-, which could be linked to a cAMP-dependent protein kinase.

Potter RL, Taylor SS
The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II.
J Biol Chem. 1980; 255: 9706-12
Display abstract
The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.

Granot J, Mildvan AS, Bramson HN, Kaiser ET
Magnetic resonance measurements of intersubstrate distances at the active site of protein kinase using substitution-inert cobalt(III) and chromium(III) complexes of adenosine 5'-(beta, gamma-methylenetriphosphate).
Biochemistry. 1980; 19: 3537-43
Display abstract
Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.

Kuroda Y, Sharma RK
Partial purification and characterization of a novel bovine adrenocortical protein kinase that phosphorylates endogenous 120,000 dalton peptide.
Biochem Biophys Res Commun. 1980; 96: 601-10

Rees DC, Lipscomb WN
Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.
Proc Natl Acad Sci U S A. 1980; 77: 4633-7
Display abstract
The structure of the complex between the proteolytic enzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the 39-amino-acid carboxypeptidase A inhibitor from potatoes has been determined at 2.5-A resolution. A combination of multiple isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging techniques was used to solve the structure. The chain trace of the inhibitor and details of the binding interactions in the complex are described. A surprising aspect of the complex is that the carboxy-terminal peptide bond of the inhibitor has been hydrolyzed, and the carboxy-terminal glycine is trapped in the binding pocket of carboxypeptidase A. Consequently, the complex resembles a stage in the catalytic mechanism after hydrolysis of the peptide bond. The ring of tyrosine-248, which is known to undergo large conformational changes upon substrate binding, is in the "down" position and interacts with the inhibitor in the complex.

Simkowski KW, Tao M
Studies on a soluble human erythrocyte protein kinase.
J Biol Chem. 1980; 255: 6456-61
Display abstract
A cyclic AMP-independent protein kinase has been isolated from human erythrocyte cytosol and purified about 28,000-fold. The enzyme uses ATP as the phosphoryl donor and exhibits a high activity towards casein and phosvitin. The kinase has a molecular weight of about 30,000 to 32,000 as estimated by sucrose density gradient centrifugation and Sephacryl S-200 gel filtration. Upon isoelectrofocusing, two kinase activities are resolved. The major component, which comprises about 85% of the activities, focuses at a pH of about 9.6 and the minor component at about 4.6. The relationship between these two kinase activities is not clear. NaF, ADP and, to a lesser extent, AMP, all inhibit the kinase activity. 2,3-Diphosphoglyceric acid, on the other hand, has no effect. The kinase also catalyzes the phosphorylation of a number of erythrocyte membrane proteins, including spectrin, band 3, and the sialoglycoproteins.

Granot J, Mildvan AS, Kaiser ET
Studies of the mechanism of action and regulation of cAMP-dependent protein kinase.
Arch Biochem Biophys. 1980; 205: 1-17

McPherson JM, Whitehouse S, Walsh DA
Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.
Biochemistry. 1979; 18: 4835-45
Display abstract
The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.

Severin ES, Sashchenko LP, Kochetkov SN, Kurochkin SN
Structure and function of protein kinase from pig brain.
Adv Cyclic Nucleotide Res. 1978; 9: 171-84

Guliaev NN, Tunitskaia VL, Baranova LA, Nesterova MV, Murtuzaev IM
[Structure of the active center of the catalytic subunit of histone kinase]
Biokhimiia. 1976; 41: 1241-9
Display abstract
A number of unknown ATP analogues is isolated when studying the structure of the active site of catalytic histonekinase subunit. Adenosine-5'-chloromethanepyrophosphonate adenosine-5'-(beta-bromoethanepyrophosphonate) and adenosine-5'-(p-fluorosulphonylphenylphosphate) were isolated under the reaction of chloromethanephosphonic acid, beta-bromoethanephosphonic acid and n-phenolsulphofluoride respectively with AMP imidazolide. Adenosine-5'-(beta-chloroethylphosphate) was obtained from AMP morpholide and ethylenechorohydrine. Adenosine-5'-chloracetylaminomethanephosphonate and adenosine-5'-(p-fluorosulphonylbenzoylaminomethanephosphonate) were obtained in the reaction of chloroacetyc anhydride and n-fluorosulphonylbenzoylchloride. Adenosine-5'-(p-aminophenylphosphate) is synthesized under the reduction of AMP mononitrophenyl ester. The treatment of the former with chloroacetyc anhydride produced adenosine-5'-(p-chloroacetylaminophenylphosphate. Interaction of ATP analogues obtained and also of early synthesized adenosine-5'-chloromethanephosphonate and adenosine-5'-(beta-bromoethanephosphonate) with homogenous catalytic histonekinase subunit is studied. The decrease in the reaction rate of Hi histone phosphorylation is found to take place. pH optimum of the enzyme inactivation with adenosine-5'-chloromethanepyrophosphonate and adenosine-5'-(beta-chloroethylphosphate) and the protective effect of the substrate (ATP) indicate covalent blocking imidazole ring in the active site. The date obtained suggest that the functional group of the active site of catalytic histonekinase subunit is histidine imidazole ring located close to terminal ATP phosphate.